Monoclonal antibodies that react with the capsule of Bacillus anthracis

ABSTRACT

The present disclosure relates to monoclonal antibodies that bind poly-γ-D-glutamic acid (γDPGA), which is present on the surface of  Bacillus anthracis . The disclosure also provides chimeric forms of the monoclonal antibodies, humanized forms of the monoclonal antibodies, and fragments thereof, as well as nucleic acids encoding the antibodies and fragments thereof. Pharmaceutical compositions including such antibodies are also disclosed herein. The disclosure further provides prophylactic, therapeutic, and diagnostic methods of using the disclosed antibodies.

REFERENCE TO RELATED APPLICATION(S)

This is a continuation of U.S. application Ser. No. 13/130,044, filedMay 18, 2011, which issued as U.S. Pat. No. 8,501,182, which is the U.S.National Stage of International Application No. PCT/US2009/065198, filedNov. 19, 2009, which was published in English under PCT Article 21(2),which in turn claims the benefit of U.S. Provisional Application No.61/116,222, filed Nov. 19, 2008. The prior applications are incorporatedherein by reference.

FIELD OF THE DISCLOSURE

This application relates to the field of antibodies, specifically tohuman antibodies that specifically bind the capsule of Bacillusanthracis and their use.

BACKGROUND

Anthrax is a potentially lethal human infection. The pathogenresponsible for anthrax is the Gram-positive rod-shaped bacterium,Bacillus anthracis (B. anthracis). In many situations, the body protectsagainst pathogenic infections by producing antibodies that bind toantigens on the pathogen to facilitate the removal or “clearance” of thepathogens by a process called phagocytosis, wherein phagocytic cells(for example neutrophils and macrophages) identify, engulf, andsubsequently destroy the pathogens. However, some pathogens, such as B.anthracis, avoid phagocytosis by encapsulating themselves with a capsulethat is poorly immunogenic and has antiphagocytic properties.

The virulence of B. anthracis is dependent on anthrax toxin (AT) and thepoly-γ-D-glutamic acid (γDPGA) capsule. γDPGA is poorly immunogenic anddoes not induce booster responses. In addition, the γDPGA capsuleshields the vegetative form of B. anthracis from agglutination bymonoclonal antibodies to its cell wall polysaccharide. Thus, feweffective antibody therapies directed against B. anthracis have beendeveloped. Accordingly, there is an ongoing need to develop therapeuticsto combat anthrax infection.

SUMMARY

Opsonins cause microorganisms to be more susceptible to phagocytosis andinterfere with the protective properties of a bacterial capsule bybinding to target antigens on the bacterial surface. This process iscalled opsonization. Opsonins, which include antibodies and complementproteins such as C3a and C5a, have lytic activities and can enhance therate of clearance of a microorganism from the bloodstream. Antibodieswhich enhance opsonophagocytosis of the microorganism Bacillus anthracisare disclosed herein.

Isolated chimpanzee monoclonal antibodies 4C and 11D, and chimeric formsthereof, humanized forms thereof, or functional fragments thereof aredisclosed. Also disclosed is a consensus anti-γDPGA chimpanzeemonoclonal antibody. The 4C and 11D monoclonal antibodies specificallybind γDPGA present on the cell surface of B. anthracis. In someexamples, the chimeric forms of these antibodies, humanized forms ofthese antibodies, and functional fragments of these antibodies includethe specificity determining regions (SDRs) and/or the complementaritydetermining regions (CDRs) of the 4C monoclonal antibody, the 11Dmonoclonal antibody or the disclosed consensus anti-γDPGA chimpanzeemonoclonal antibody. The monoclonal antibodies, chimeric forms,humanized forms or functional fragments thereof can be conjugated to aneffector molecule, such as a detectable marker, a therapeutic agent, ora toxin. Also disclosed are nucleic acid molecules encoding thesedisclosed antibodies. In addition, kits for detecting B. anthracis in asample are disclosed herein.

Methods are disclosed for detecting B. anthracis. Such methods includecontacting a biological sample with one of the disclosed antibodies, achimeric form thereof, a humanized form thereof, or a functionalfragment thereof, under conditions wherein an immune complex will form,and detecting the formation of the immune complex. The detection of animmune complex indicates the presence of B. anthracis.

Methods for treating or inhibiting B. anthracis infection are alsodisclosed. The disclosed methods include administering to a subject aneffective amount of one or more of the disclosed antibodies, a chimericform thereof, a humanized form thereof, or a functional fragmentthereof, thereby inhibiting the B. anthracis infection.

Also provided herein are methods of enhancing opsonophagocytosis of B.anthracis in a subject. The method includes selecting for treatment asubject who is at risk for developing a B. anthracis infection or isinfected by B. anthracis and administering a therapeutically effectiveamount of one or more of the disclosed antibodies, a chimeric formthereof, a humanized form thereof, or a functional fragment thereof,thereby enhancing the opsonophagocytosis of B. anthracis.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is an alignment of the amino acid sequence of the heavy chain ofthe anti-γDPGA monoclonal antibodies (mAbs) 4C and 11D. The figure showsthe alignment of the sequence of the 4C antibody heavy chain (top; SEQID NO: 3) with the sequence of the 11D antibody heavy chain (below; SEQID NO: 5). Identical residues are identified by a dot (.); missingresidues are identified by a dash (-). The location of the frameworkregions and the complementarity determining regions (CDRs) are alsoshown.

FIG. 1B is an alignment of the amino acid sequence of the light chain ofthe anti-γDPGA monoclonal antibodies 4C and 11D. The figure shows thealignment of the sequence of the 4C antibody light chain (top; SEQ IDNO: 4) with the sequence of the 11D antibody light chain (below; SEQ IDNO: 6). Identical residues are identified by a dot (.); missing residuesare identified by a dash (-). The location of the framework regions andthe complementarity determining regions (CDRs) are also shown.

FIG. 2 is as a graph of an exemplary binding curve of the 4C and 11Danti-γDPGA monoclonal antibodies converted to full-length IgG with humanγ1 constant regions, showing the binding of these antibodies to γDPGA asmeasured by an enzyme-linked immunosorbent assay (ELISA).

FIG. 3 is a set of digital images of a capsular quellung type reactionusing the 4C monoclonal antibody. Cells of formalin-killed B. anthracis(Ames 34 strain) were incubated with IgG1 of anti-γDPGA 4C monoclonalantibody at concentrations of 25 μg/ml (FIG. 3C), 50 μg/ml (FIG. 3D),and 100 μg/ml (FIG. 3E). The Fab of anti-γDPGA ml (FIG. 3B) or IgG1 ofanti-PA ml (FIG. 3A) at 100 μg/ml were used as controls. The reactionswere assessed by DIC microscopy and show that IgG1 of anti-γDPGA atconcentrations of 50 μg/ml and 100 μg/ml produced a rim type reaction atthe capsule perimeter of formalin-killed B. anthracis (Ames 34 strain).

FIG. 4 is a set of graphs showing the opsonophagocytic activity ofanti-γDPGA monoclonal antibodies 4C (FIG. 4A) and 11D (FIG. 4B) asmeasured by their ability to kill B. anthracis cells in the presence ofhuman polymorphonuclear leukocytes and complement. Opsonophagocytosiswas defined as ≧50% killing compared with growth in control (noantibody) wells.

FIG. 5 is a graph showing the effect of IgG1 and IgG3 isotypes of the 4Cand 11D monoclonal antibodies on the survival of mice exposed tovirulent anthrax spores.

Biological Deposit

Plasmids containing DNA encoding the monoclonal antibodies 4C and 11Dwere deposited in accordance with the Budapest Treaty with the AmericanType Culture Collection (ATCC) Patent Depository, 10801 UniversityBlvd., Manassas, Va., 20110, on Nov. 14, 2008. The plasmid pComb3H-4Cencoding the 4C FAb sequence was deposited as Accession No. PTA-9610.The plasmid pComb3H-11D encoding the 11D Fab sequence was deposited asAccession Nos. PTA-9609.

Sequence Listing

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

SEQ ID NO: 1 is the consensus sequence of an anti-γDPGA antibody heavychain.

SEQ ID NO: 2 is the consensus sequence of an anti-γDPGA antibody lightchain.

SEQ ID NO: 3 is an exemplary amino acid sequence of a 4C monoclonalantibody heavy chain.

SEQ ID NO: 4 is an exemplary amino acid sequence of a 4C monoclonalantibody light chain.

SEQ ID NO: 5 is an exemplary amino acid sequence of an 11D monoclonalantibody heavy chain.

SEQ ID NO: 6 is an exemplary amino acid sequence of an 11D monoclonalantibody light chain.

SEQ ID NO: 7 is an exemplary nucleic acid sequence encoding a 4Cmonoclonal antibody heavy chain.

SEQ ID NO: 8 is an exemplary nucleic acid sequence encoding a 4Cmonoclonal antibody light chain.

SEQ ID NO: 9 is an exemplary nucleic acid sequence encoding an 11Dmonoclonal antibody heavy chain.

SEQ ID NO: 10 is an exemplary nucleic acid sequence encoding an 11Dmonoclonal antibody light chain.

SEQ ID NOs: 11-18 are exemplary amino acid sequences of human frameworkregions.

DETAILED DESCRIPTION

I. Abbreviations

AT Anthrax toxin

ATR Anthrax toxin receptor

CDR Complementarity determining region

C_(H) Heavy chain constant region

C_(L) Light chain constant region

EF Edema factor

EM Effector moiety/molecule

ELISA Enzyme linked immunosorbant assay

Fab Fragment, antigen binding

FACS Fluorescence activated cell sorting

Fc Fragment, crystallizable

FITC fluorescein isothiocyanate

γ-DPGA Poly-γ-D-glutamic acid

HRP Horseradish peroxidase

LF Lethal factor

LeTx Lethal toxin

mAb Monoclonal antibody

PA Protective antigen

PE Phycoerythrin

PNAs Peptide nucleic acids

RIA Radioimmunoassay

SDR Specificity determining region

T Threonine

V Valinr

VH Variable heavy

VL Variable light

YFP Yellow fluorescent protein

II. Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

As used herein, the singular forms “a,” “an,” and “the,” refer to boththe singular as well as plural, unless the context clearly indicatesotherwise. For example, the term “antibody” includes single or pluralantibodies and can be considered equivalent to the phrase “at least oneantibody.”

As used herein, the term “comprises” means “includes.” Thus, “comprisingan antibody” means “including an antibody” without excluding otherelements.

It is further to be understood that all base sizes or amino acid sizes,and all molecular weight or molecular mass values, given for nucleicacids or polypeptides are approximate, and are provided for descriptivepurposes, unless otherwise indicated. Although many methods andmaterials similar or equivalent to those described herein can be used,particular suitable methods and materials are described below. In caseof conflict, the present specification, including explanations of terms,will control. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

To facilitate review of the various embodiments of the invention, thefollowing explanations of terms are provided:

Administration: The introduction of a composition into a subject by achosen route. For example, if the chosen route is intravenous, thecomposition is administered by introducing the composition into a veinof the subject. In some examples an antibody or antibody fragment, suchas those described herein, is administered.

Amplification: Refers to use of a technique that increases the number ofcopies of a nucleic acid molecule in a sample, for example theamplification of a nucleic acid that encodes the monoclonal antibody 4Cor 11D, a chimeric form thereof, a humanized form thereof, or a fragmentthereof. An example of amplification is the polymerase chain reaction,in which a biological sample collected from a subject is contacted witha pair of oligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in PCT Publication No. WO 90/01069; ligase chain reactionamplification, as disclosed in European patent publication No. EP-A-320308; gap filling ligase chain reaction amplification, as disclosed inU.S. Pat. No. 5,427,930; and NASBA™ RNA transcription-freeamplification, as disclosed in U.S. Pat. No. 6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen or a fragment thereof. Antibodies canbe composed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (V_(H)) region and the variable light(V_(L)) region. Together, the V_(H) region and the V_(L) region areresponsible for binding the antigen recognized by the antibody.

The term antibody includes intact immunoglobulins and the variants andportions of them well known in the art, such as Fab′ fragments, F(ab)′2fragments, single chain Fv proteins (“scFv”), and disulfide stabilizedFv proteins (“dsFv”). A scFv protein is a fusion protein in which alight chain variable region of an immunoglobulin and a heavy chainvariable region of an immunoglobulin are bound by a linker, while indsFvs, the chains have been mutated to introduce a disulfide bond tostabilize the association of the chains. The term also includesgenetically engineered forms such as chimeric antibodies (for example,an antibody with non-human variable regions and human constant regionsor with a non-human Fab region and a human Fc region), humanizedantibodies (for example, an antibody with non-human SDRs and/or CDRs andhuman framework regions), heteroconjugate antibodies (such as,bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995(Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed.,W.H. Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds an antigen of interest hasa specific V_(H) region and the V_(L) region sequence, and thus specificCDR sequences. Antibodies with different specificities (due to differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone of ahybridoma generated from a B-lymphocyte and a myeloma cell, or by a celltransfected with the light and heavy chain genes of a single antibody,or by the progeny thereof. The preparation of monoclonal antibodies isconventional. See, for example, Kohler & Milstein, Nature 256:495, 1975;Coligan et al., sections 2.5.1-2.6.7; and Harlow et al., Antibodies: ALaboratory Manual, p. 726, Cold Spring Harbor Pub., 1988. Briefly,monoclonal antibodies can be obtained by injecting mice with acomposition comprising an antigen, verifying the presence of antibodyproduction by removing a serum sample, removing the spleen to obtain Blymphocytes, fusing the B lymphocytes with myeloma cells to producehybridomas, cloning the hybridomas, selecting positive clones thatproduce antibodies to the antigen, and isolating the antibodies from thehybridoma cultures. Monoclonal antibodies can be isolated and purifiedfrom hybridoma cultures by a variety of well-established techniques.Hybridoma cells, and their progeny, that secrete monoclonal antibodiesare also encompassed by this disclosure. Monoclonal antibodies includechimpanzee, human, and humanized monoclonal antibodies, such ashumanized chimpanzee monoclonal antibodies. In some examples, amonoclonal antibody is the monoclonal antibody 4C or 11D or a consensusanti-γDPGA monoclonal antibody.

A “consensus monoclonal antibody” has a consensus light chain amino acidsequence and a consensus heavy chain amino acid sequence, wherein eachconsensus amino acid sequence represents the results of multiplesequence alignments of related sequences and the consensus sequenceshows amino acid residues that are common among the aligned sequences.Common residues in the consensus light chain and heavy chain sequencesare conserved amino acids and are generally important for antigenbinding. The residues that vary among the aligned sequences representamino acid positions that can tolerate sequence variability and aregenerally not important for antigen binding.

A “chimeric antibody” is an antibody that includes sequences derivedfrom two different antibodies, which typically are of different species.Chimeric antibodies generally include human constant regions andvariable regions from other animal sources, such as chimpanzees, forexample chimpanzee Fab regions, CDRs, and/or chimpanzee SDRs. In someexamples, a chimeric antibody includes the Fab region from a chimpanzeeantibody and an Fc region from a human antibody. In particular examplesa chimeric antibody includes the SDRs or CDRs from a chimpanzeeantibody.

A “humanized” antibody immunoglobulin is an immunoglobulin including ahuman variable framework region and one or more CDRs and/or SDRs from anon-human (for example a chimpanzee, mouse, rat, or synthetic)immunoglobulin. The non-human immunoglobulin providing the CDRs or SDRsis termed a “donor,” and the human immunoglobulin providing the variableframework region is termed an “acceptor.” In one embodiment, all theCDRs are from the donor immunoglobulin in a humanized antibody. Inanother embodiment, only SDRs are from the donor immunoglobulin in ahumanized antibody. Constant regions need not be present, but if theyare, they must be substantially identical to human immunoglobulinconstant regions, such as at least about 85-90%, such as about 95% ormore identical. Hence, in one embodiment, all parts of a humanizedimmunoglobulin, except possibly one or more CDRs and/or SDRs, aresubstantially identical to corresponding parts of natural humanimmunoglobulin sequences. In one embodiment, a “humanized antibody” isan antibody comprising a humanized light chain and a humanized heavychain immunoglobulin. A humanized antibody binds to the same antigen asthe donor antibody that provides the CDRs or SDRs. The acceptorframework of a humanized immunoglobulin or antibody may have a limitednumber of substitutions by amino acids taken from the donor framework.Humanized or other monoclonal antibodies can have additionalconservative amino acid substitutions which have substantially no effecton antigen binding or other immunoglobulin functions. Humanizedimmunoglobulins can be constructed by means of genetic engineering (seefor example, U.S. Pat. No. 5,585,089).

Bacillus: A genus of bacteria whose characteristics include the abilityto degrade of most substrates derived from plant and animal sources,including cellulose, starch, pectin, proteins, agar, hydrocarbons, andothers; antibiotic production; nitrification; denitrification; nitrogenfixation; facultative lithotrophy; autotrophy; acidophily; alkaliphily;psychrophily, thermophily and parasitism. Spore formation, a universalcharacteristic of the genus, is thought to be a strategy for Bacillussurvival in the soil environment, wherein this genus of bacteriapredominate. Aerial distribution of dormant spores likely explains theoccurrence of Bacillus species in most habitats examined.

There are more than 40 recognized species in the genus Bacillus(Bergey's Manual of Systematic Bacteriology Vol. 2 (1986)). Theseinclude, but are not limited to, B. acidocaldarius, B. alkalophilus, B.alvei, B. anthracis, B. azotoformans, B. badius, B. brevis, B. cereus,B. circulans, B. coagulans, B. fastidiosis, B. firmus, B. globisporus,B. insolitus, B. larvae, B. laterosporus, B. lentimorbus, B. lentus, B.licheniformis, B. macerans, B. macquariensis, B. marinus, B. megaterium,B. mycoides, B. pantothenticus, B. pasteurii, B. polymyxa, B. popillia,B. pumilus, B. schlegelii, B. sphaericus, B. stearothermophilus, B.subtilis, and B. thuringiensis. In one specific, non-limiting example, aBacillus is Bacillus anthracis, the agent that causes anthrax.

Bacillus Anthracis: The etiologic agent of anthrax, Bacillus anthracisis a large, gram-positive, nonmotile, spore-forming bacterial rod. Thevirulence of B. anthracis is dependent on anthrax toxin (AT), and theγDPGA capsule. The genes for the toxin, and the capsule, are carried byplasmids, designated pX01 and pX02, respectively (Mikesell et al.,Infect. Immun. 39:371-76, 1983; Vodkin et al., Cell 34:693-97, 1983;Green et al., Infect. Immun. 49:291-97, 1985).

AT is composed of three entities: protective antigen (PA; the bindingsubunit of AT), and two enzymes known as lethal factor (LF) and edemafactor (EF) (Mikesell et al., Infect. Immun. 39:371-76, 1983; Vodkin etal., Cell 34:693-97, 1983). PA is an 83 kDa protein that is the mainprotective constituent of anthrax vaccines. PA binds to the anthraxtoxin receptor (ATR) on cells and is then proteolytically cleaved by theenzyme furin with release of a 20 kDa fragment (Bradley et al., Nature414:225-29, 2001; Klimpel et al., PNAS 89:10277-81, 1992). The 63 kDa PAremnant (PA₆₃) features a second binding domain and binds to either EF(an 89 kDa protein) to form edema toxin, or LF (a 90 kDa protein) toform lethal toxin (LeTx) (Leppla et al., Salisbury Med. Bull. Suppl.68:41-43, 1990). The resulting complex is internalized into the cellwithin endosomes (Singh et al., Infect. Immun. 67:1853-59, 1999;Friedlander, J. Biol. Chem. 261:7123-26, 1986).

The γDPGA capsule of B. anthracis serves as an essential virulencefactor during anthrax infection, inhibiting host defense mechanismsthrough inhibition of phagocytosis of microorganism by macrophages.While other Bacillus produce γPGA in a mixture of both D- and L-forms,only B. anthracis is known to synthesize it exclusively in aD-conformation (Kovács et al., J. Chem. Soc. 4255-59, 1952). Wheninjected, γDPGA has been shown to be a poor immunogen (Eisner, Schweiz.Z. Pathol. Bakteriol. 22:129-44, 1959; Ostroff et al., Proc. Soc. Exp.Biol. Med. 99:345-47, 1958). The capsule also shields the vegetativeform of B. anthracis from agglutination by monoclonal antibodies to itscell wall polysaccharide (Ezzell et al., J. Clin. Microbiol. 28:223-31,1990).

Binding affinity: Affinity of an antibody, such as the monoclonalantibody 4C or 11D, for an antigen, such as an antigen on the surface ofB. anthracis for example γDPGA. In one embodiment, affinity iscalculated by a modification of the Scatchard method described byFrankel et al., Mol. Immunol., 16:101-106, 1979. In another embodiment,binding affinity is measured by an antigen/antibody dissociation rate.In yet another embodiment, a high binding affinity is measured by acompetition radioimmunoassay. In several examples, a high bindingaffinity is at least about 1×10⁻⁸ M. In other embodiments, a highbinding affinity is at least about 1.5×10⁻⁸ M, at least about 2.0×10⁻⁸M, at least about 2.5×10⁻⁸ M, at least about 3.0×10⁻⁸ M, at least about3.5×10⁻⁸ M, at least about 4.0×10⁻⁸ M, at least about 4.5×10⁻⁸ M, or atleast about 5.0×10⁻⁸ M.

Complement: A plasma protein system involved in immune defense.Following activation by antigen-antibody complexes, complement proteinslyse antigenic cells, attract phagocytic cells, and assist in thedestruction of antigenic cells by opsonophagocytosis. In mammals, thecomplement system is made up of a series of about 25 proteins that workto “complement” the activity of antibodies in destroying bacteria,either by facilitating opsonophagocytosis or by puncturing the bacterialcell membrane. Complement also helps to rid the body of antigen-opsonicantibody complexes, for example by clearance of a pathogen that is boundby the opsonic antibody.

Complement proteins circulate in the blood in an inactive form until thefirst of the complement substances is triggered—usually by an antibodyinterlocked with an antigen. As each complement protein is activated, itacts upon another complement protein in a precise sequence of carefullyregulated steps known as the “complement cascade.”

Complement fragments (such as C3a, C3b, iC3b, C3d, C4b, or C5a, whichbecome bound to antigen during complement activation) triggeropsonophagocytosis by binding to specific cell-surface receptors (suchas Fc receptors and C3b receptors on neutrophils and macrophages, andC3d receptors on macrophages).

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of animmunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3and H-CDR1, H-CDR2, H-CDR3, respectively. By definition, the CDRs of thelight chain are bounded by the residues at positions 24 and 34 (L-CDR1),50 and 56 (L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain arebounded by the residues at positions 31 and 35b (H-CDR1), 50 and 65(H-CDR2), 95 and 102 (H-CDR3), using the numbering convention delineatedby Kabat et al., (1991) Sequences of Proteins of Immunological Interest,5^(th) Edition, U.S. Department of Health and Human Services, PublicHealth Service, National Institutes of Health, Bethesda, Md. (NIHPublication No. 91-3242). CDRs contain the specificity determiningregions (SDRs) of the antibody. In some examples, a CDR is a CDR fromthe monoclonal antibody 4C. In some examples, a CDR is a CDR from themonoclonal antibody 11D. In some examples, a CDR is a CDR from theconsensus anti-γDPGA chimpanzee monoclonal antibody.

Conservative variants: Amino acid substitutions that do notsubstantially affect or decrease the affinity of an antibody to B.anthracis capsule. For example, an antibody that specifically binds B.anthracis capsule (such as the antibodies 4C or 11D, the consensusanti-γDPGA chimpanzee monoclonal antibody a chimeric form, or humanizedform or a fragment thereof) can include at most about 1, at most about2, at most about 5, and most about 10, or at most about 15 conservativesubstitutions and specifically bind the original anthrax capsule. Theterm conservative variation also includes the use of a substituted aminoacid in place of an unsubstituted parent amino acid, provided thatantibody specifically binds B. anthracis capsule.

Conservative amino acid substitution tables providing functionallysimilar amino acids are well known to one of ordinary skill in the art.The following six groups are examples of amino acids that are consideredto be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Contacting: Placement in direct physical association. Such physicalassociations can be between solids, liquids, or solids and liquids.

Degenerate variant: A polynucleotide encoding an antibody that bindsanthrax capsule that includes a sequence that is degenerate as a resultof the genetic code. There are 20 natural amino acids, most of which arespecified by more than one codon. Therefore, all degenerate nucleotidesequences are included as long as the amino acid sequence of theantibody that binds B. anthracis capsule encoded by the nucleotidesequence is unchanged.

Effective amount or Therapeutically effective amount: The amount ofagent, such as the disclosed antibodies, the chimeric forms thereof, thehumanized forms thereof, or the antibody fragments thereof, that is anamount sufficient to prevent (including prophylaxis), treat, reduce,and/or ameliorate the symptoms and/or underlying causes of any of adisorder or disease, for example to prevent, inhibit, and/or treat aninfection with B. anthracis. In some embodiments, an “effective amount”or a “therapeutically effective amount” is an amount sufficient toreduce or eliminate a symptom of a disease, such as anthrax.

Effector cells: In one embodiment, effector cells are cells capable ofbinding to antibody/antigen complexes and internalizing such complexes.The complexes can be bound to a cell, microbe, or pathogen such that thecell, microbe, or pathogen is also internalized by the effector cell. Inparticular examples, effector cells express Fc receptors, such as FcγRI,FcγRII and FcγRIII that bind to antibody/antigen complexes andfacilitate internalization. In some examples, effector cells are derivedfrom the serum of an individual (such as peripheral blood leukocytes,PBLs) or from an in vitro culture. Examples of effector cells include,but are not limited to: macrophages, mononuclear phagocytes, naturalkiller cells, and granulocytes such as neutrophils and eosinophils.

Effector molecule: The portion of a molecule, for example a chimericmolecule that includes a disclosed antibody or fragment thereof, that isintended to have a desired effect on a cell, such as a B. anthraciscell, to which the molecule is targeted. Effector molecules are alsoknown as an effector moieties (EM), therapeutic agents, or diagnosticagents, or similar terms. In some embodiments, effector molecules can bediagnostic agents or moieties. Diagnostic agents or moieties includeradioisotopes and other detectable labels. Detectable labels useful forsuch purposes are also well known in the art, and include radioactiveisotopes such as ³²P, ¹²⁵I, and ¹³¹I, fluorophores, chemiluminescentagents, magnetic resonance imaging agents and enzymes.

Epitope: An antigenic determinant, such as that on the capsule of B.anthracis. Epitopes or antigenic determinants are particular chemicalgroups or peptide sequences on a molecule that are antigenic and canelicit a specific immune response. An antibody specifically binds aparticular antigenic epitope.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium. In some examples, a disclosed antibody or fragment thereof isexpressed from a nucleic acid sequence, for example expressed from anexpression vector.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence, for example the transcription and translation ofthe nucleic acid sequence encoding a disclosed antibody or fragmentthereof from an expression vector, for example from a host celltransformed with an expression vector. Expression control sequences caninclude appropriate promoters, enhancers, transcription terminators, astart codon (i.e., ATG) in front of a protein-encoding gene, splicingsignal for introns, and maintenance of the correct reading frame of thatgene to permit proper translation of mRNA, and stop codons. The term“control sequences” is intended to include, at a minimum, componentswhose presence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, pap, ptac (ptrp-lac hybrid promoter) and thelike may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Framework Region: Amino acid sequences interposed between CDRs, andincludes variable light and variable heavy framework regions. Theframework regions serve to hold the CDRs of an antibody or fragmentthereof in an appropriate orientation for antigen binding. In someexamples, a framework region is a human framework region. In someexamples, a framework region is a chimpanzee framework region, such asframework region from the 4C or 11D monoclonal antibody or from theconsensus anti-γDPGA chimpanzee monoclonal antibody.

Heterologous: A heterologous sequence is a sequence that is not normallyfound adjacent to a second sequence. In one embodiment, the heterologoussequence is from a different genetic source, such as a virus or adifferent organism, than the second sequence. For example a nucleotidesequence encoding a 4C or 11D antibody or the consensus anti-γDPGAchimpanzee monoclonal antibody can be operably linked to a heterologousnucleotide sequence encoding a fluorescent molecule capable ofdetection, such as a fluorescent protein, for example green fluorescentprotein (GFP).

Host cells: Cells in which a vector can be propagated and its DNAexpressed, for example a vector encoding a disclosed antibody offragment thereof. The cell may be prokaryotic or eukaryotic. The termalso includes any progeny of the subject host cell. It is understoodthat all progeny may not be identical to the parental cell since theremay be mutations that occur during replication. However, such progenyare included when the term “host cell” is used. In some examples, a hostcell propagates a vector encoding a 4C or 11D antibody, the consensusanti-γDPGA chimpanzee monoclonal antibody, humanized form thereof, orchimeric form thereof, or functional fragment thereof.

Immunoconjugate: A covalent linkage of an effector molecule to anantibody, such as a 4C or 11D antibody, the consensus anti-γDPGAchimpanzee monoclonal antibody, humanized form thereof, or chimeric formthereof, or functional fragment thereof. In some examples the effectormolecule can be a detectable label. In one embodiment, an antibodylinked (coupled) to an effector molecule is further linked to a lipid orother molecule to a protein or peptide to increase its half-life in thebody. The linkage can be achieved either by chemical or recombinantmeans. When the linkage is chemical, a reaction between the antibodymoiety and the effector molecule has produced a covalent bond formedbetween the two molecules to form one molecule. A peptide linker (shortpeptide sequence) can optionally be included between the antibody andthe effector molecule. Because immunoconjugates are prepared from twomolecules with separate functionalities, such as an antibody and aneffector molecule, they are also sometimes referred to as “chimericmolecules.” The term “chimeric molecule,” as used with reference to aneffector molecule, therefore refers to a targeting moiety, such as aligand or an antibody, conjugated (coupled or covalently linked) to aneffector molecule.

Immunologically reactive conditions: Conditions in which an antibodyraised against a particular epitope bind to that epitope (or to a cellexpressing the epitope) to a detectably greater degree than, and/or tothe substantial exclusion of, binding to substantially all otherepitopes (or cells not expressing the epitope). Immunologically reactiveconditions are dependent upon the format of the antibody bindingreaction and typically are those utilized in immunoassay protocols orthose conditions encountered in vivo. See Harlow & Lane, supra, for adescription of immunoassay formats and conditions. The immunologicallyreactive conditions employed in the methods are “physiologicalconditions” which include reference to conditions (such as temperature,osmolarity, pH) that are typical inside a living mammal or a mammaliancell. While it is recognized that some organs are subject to extremeconditions, the intra-organismal and intracellular environment normallylies around pH 7 (i.e., from pH 6.0 to pH 8.0, more typically pH 6.5 to7.5), contains water as the predominant solvent, and exists at atemperature above 0° C. and below 50° C. Osmolarity is within the rangethat is supportive of cell viability and proliferation.

Inhibiting or treating a pathological condition or disease: Inhibitingthe full or partial development of a disease or pathological condition,for example, in a subject who is at risk at being infected by Bacillusanthracis or who is at risk for a disease such as anthrax. “Treatment”refers to a therapeutic intervention that ameliorates a sign or symptomof a disease or pathological condition after it has begun to develop.The term “ameliorating,” with reference to a disease or pathologicalcondition, refers to any observable beneficial effect of the treatment.The beneficial effect can be evidenced, for example, by a delayed onsetof clinical symptoms of the disease or condition in a susceptiblesubject, a reduction in severity of some or all clinical symptoms of thedisease or condition, a slower progression of the disease or condition,an improvement in the overall health or well-being of the subject, or byother parameters well known in the art that are specific to theparticular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orcondition, or exhibits only early signs of such. In one embodiment, aprophylactic treatment is administered for the purpose of decreasing therisk of developing a pathology associated with a disease or condition.

Isolated: An “isolated” biological component (such as a nucleic acid,polypeptide, for example and antibody or fragment thereof (for example4C, 11D antibody, or the consensus anti-γDPGA chimpanzee monoclonalantibody, a humanized form thereof, or a chimeric form thereof, or afunctional fragment thereof), cell or protein) that has beensubstantially separated, produced apart from, or purified away fromother biological components in the cell of the organism in which thecomponent naturally occurs. Nucleic acids and polypeptides which havebeen “isolated” thus include nucleic acids and polypeptides purified bystandard purification methods. The term also embraces nucleic acids andpolypeptides prepared by recombinant expression in a host cell as wellas chemically synthesized nucleic acids. A purified nucleic acid,polypeptide, cell, or cell component can be at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% pure.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody (for example a 4Cor 11D antibody, a consensus anti-γDPGA chimpanzee monoclonal antibody,humanized form thereof, or chimeric form thereof, or functional fragmentthereof) or a protein, to facilitate detection of that molecule.Specific, non-limiting examples of labels include detectable markers,such as fluorescent tags, enzymatic linkages, and radioactive isotopes.In specific, non-limiting embodiments, an amino acid is radiolabeled ora polypeptide is conjugated to biotinyl moieties that can be detected bymarked avidin (for example, streptavidin containing a fluorescent markeror an enzymatic activity that can be detected by optical or colorimetricmethods). Various methods of labeling polypeptides and glycoproteins areknown in the art and may be used. Examples of labels for polypeptidesinclude, but are not limited to, the following: radioisotopes orradionuclides (such as ³⁵S or ¹³¹I), fluorescent labels (such asfluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors,fluorescent proteins, such as green fluorescent protein), enzymaticlabels (such as horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase), chemiluminescent markers, biotinyl groups,predetermined polypeptide epitopes recognized by a secondary reporter(such as a leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags), or magnetic agents,such as gadolinium chelates. In some embodiments, labels are attached byspacer arms or linkers of various lengths to reduce potential sterichindrance.

Linker peptide: A peptide within an antibody binding fragment (such asan Fv fragment, for example a 4C or 11D antibody fragment) which servesto indirectly bond the variable heavy chain to the variable light chain.“Linker” can also refer to a peptide serving to link a targeting moiety,such as a scFv, to an effector molecule, such as a detectable label.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an antibody. In the specific context, the terms includereference to joining a molecule, such as an antibody moiety, to aneffector molecule (“EM”). The linkage can be either by chemical orrecombinant means. “Chemical means” refers to a chemical reactionbetween the antibody moiety and the effector molecule such that there isa covalent bond formed between the two molecules to form one molecule.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Non-naturallyoccurring synthetic analogs include, for example and without limitation,phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs),and the like. Such polynucleotides can be synthesized, for example,using an automated DNA synthesizer. The term “oligonucleotide” typicallyrefers to short polynucleotides, generally no greater than about 50nucleotides. It will be understood that when a nucleotide sequence isrepresented by a DNA sequence (i.e., A, T, G, C), this also includes anRNA sequence (i.e., A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom, for example an antibody, suchas a 4C or 11D antibody, a consensus anti-DPGA chimpanzee monoclonalantibody, or a portion of an antibody, such as V_(H) or V_(L) from a 4Cor 11D antibody. Thus, a gene encodes a protein if transcription andtranslation of mRNA produced by that gene produces the protein in a cellor other biological system. Both the coding strand (the nucleotidesequence of which is identical to the mRNA sequence and is usuallyprovided in sequence listings) and non-coding strand (used as thetemplate for transcription) of a gene or cDNA can be referred to asencoding the protein or other product of that gene or cDNA. Unlessotherwise specified, a “nucleotide sequence encoding an amino acidsequence” includes all nucleotide sequences that are degenerate versionsof each other and that encode the same amino acid sequence. Nucleotidesequences that encode proteins and RNA may include introns.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence (for example a coding sequence of an antibody or fragmenttherefore herein disclosed) if the promoter affects the transcription orexpression of the coding sequence. Generally, operably linked DNAsequences are contiguous and, where necessary to join two protein codingregions, in the same reading frame.

Opsonin: A molecule that becomes attached to the surface of a microbe orpathogen, such as a bacterial, fungal, or viral pathogen, that can berecognized by surface receptors of neutrophils and macrophages and thatincreases the efficiency of phagocytosis of the microbe or pathogen.Opsonins include IgG antibodies, which are recognized by the Fcγreceptor on phagocytes, and fragments of complement proteins, which arerecognized by the cell surface protein CR1 (CD35) and by the leukocyteintegrin Mac-1. Exemplary opsonins include opsonizing antibodies (IgM,IgG1, IgG2, IgG3 and IgA immunoglobulins specific for the antigen), suchas a 4C or 11D antibody, humanized form thereof, or chimeric formthereof, or functional fragment thereof, and certain complementfragments (C3a, C3b, iC3b, C3d, C4b, or C5a, which become bound to theantigen during complement activation), both of which triggerphagocytosis by binding to specific cell-surface receptors (such as Fcreceptors and C3b receptors on neutrophils and macrophages, and C3dreceptors on macrophages). Opsonins include any substance that binds toparticulate antigens on the surface of a cell and induces thephagocytosis of the cell by effector cells.

Opsonophagocytosis (opsonization): The process of enhancing the abilityof effector cells (such as macrophages and monocytes) to targetmicroorganisms for phagocytosis, by recognizing opsonins (for exampleantibodies or complement proteins) attached to the surface of themicroorganism.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide. In some examples an openreading frame encodes an antibody or antibody fragment, such as thosedisclosed herein.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds, for example γ amide bonds(for example from the γ position of a glutamic acid side chain) or aamide bonds. When the amino acids are alpha-amino acids, either theL-optical isomer or the D-optical isomer can be used, for exampleD-glutamic acid to form poly-γ-D-glutamic acid (γDPGA). The terms“polypeptide” or “protein” as used herein is intended to encompass anyamino acid sequence and include modified sequences such asglycoproteins. The term “polypeptide” is specifically intended to covernaturally occurring proteins, as well as those that are recombinantly orsynthetically produced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide. Biologically functionalfragments, for example, can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in conjunction with the antibodies and fragments thereofdisclosed herein are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the antibodies and antibody fragments herein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical or therapeutic agent: A composition capable of inducing adesired therapeutic or prophylactic effect when properly administered toa subject or a cell. In some embodiments, a pharmaceutical ortherapeutic agent is an antibody, or fragment thereof disclosed herein.In one specific non-limiting example, a pharmaceutical agent is anantibody that specifically binds B. anthracis.

Poly-γ-D-glutamic acid (γDPGA): A homopolymer of glutamic acid residueslinked by γ peptide bonds. The glutamic acid residues constituting thehomopolymer are be solely in the D-form (γDPGA).

Purified: The term purified does not require absolute purity; rather, itis intended as a relative term. Thus, for example, a purified peptidepreparation is one in which the peptide or protein is more enriched thanthe peptide or protein is in its natural environment. In one embodiment,a preparation is purified such that the protein or peptide represents atleast 50% of the total peptide or protein content of the preparation.

Substantial purification denotes purification from other proteins orcellular components. A substantially purified protein is at least 60%,70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limitingexample, a substantially purified protein is 90% free of other proteinsor cellular components. The disclosed antibodies or fragments thereofthat specifically bind B. anthracis capsule, can be purified by any ofthe means known in the art. See for example Guide to ProteinPurification, ed. Deutscher, Meth. Enzymol. 185, Academic Press, SanDiego, 1990; and Scopes, Protein Purification: Principles and Practice,Springer Verlag, New York, 1982.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, such as by genetic engineering techniques. Similarly,a recombinant protein is one encoded for by a recombinant nucleic acidmolecule.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity (or similarity or homology);the higher the percentage, the more similar the two sequences are.Homologs or variants of a 4C or 11D antibody, the consensus anti-γDPGAchimpanzee monoclonal antibody, humanized form thereof, or chimeric formthereof, or functional fragment thereof will possess a relatively highdegree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody thatspecifically binds Bacillus anthracis capsule are typicallycharacterized by possession of at least about 75%, for example at leastabout 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted overthe full length alignment with the amino acid sequence of the antibodyusing the NCBI Blast 2.0, gapped blastp set to default parameters. Forcomparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and may possess sequence identities of at least 85% or at least90% or 95% depending on their similarity to the reference sequence.Methods for determining sequence identity over such short windows areavailable at the NCBI website on the internet. One of skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. In several embodiments, a specific binding agent is themonoclonal 4C or 11D antibody, the consensus anti-γDPGA chimpanzeemonoclonal antibody humanized form thereof, chimeric form thereof, orfunctional fragment thereof. The term “specifically binds” refers to thepreferential association of an antibody or other ligand, in whole orpart, with that antigen or cells bearing that antigen, and not to otherantigens or cells or tissues lacking that antigen. It is, of course,recognized that a certain degree of non-specific interaction may occurbetween a molecule and a non-target cell. Nevertheless, specific bindingmay be distinguished as mediated through specific recognition of theantigen. Although selectively reactive antibodies bind antigen, they maydo so with low affinity. On the other hand, specific binding results ina much stronger association between the antibody or other specificbinding agent and the antigen or cells bearing the antigen, than betweenthe bound antibody (or other specific binding agent) and cells lackingthe antigen. Specific binding typically results in greater than 2-fold,such as greater than 5-fold, greater than 10-fold, or greater than100-fold increase in amount of bound antibody or other ligand (per unittime) to a cell or tissue expressing the target epitope as compared to acell or tissue lacking this epitope. A variety of immunoassay formatsare appropriate for selecting antibodies or other ligands specificallyimmunoreactive with a particular protein. For example, solid-phase ELISAimmunoassays are routinely used to select monoclonal antibodiesspecifically immunoreactive with a protein. See Harlow & Lane,Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NewYork (1988), for a description of immunoassay formats and conditionsthat can be used to determine specific immunoreactivity.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” or“transfected” by a nucleic acid introduced into a cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication.

Numerous methods of transfection are known to those skilled in the art,such as: chemical methods (for example, calcium-phosphate transfection),physical methods (for example, electroporation, microinjection, particlebombardment), fusion (for example, liposomes), receptor-mediatedendocytosis (for example, DNA-protein complexes, viralenvelope/capsid-DNA complexes) and by biological infection by virusessuch as recombinant viruses (see, for example, Wolff, J. A., ed, GeneTherapeutics, Birkhauser, Boston, USA (1994)). In the case of infectionby retroviruses, the infecting retrovirus particles are absorbed by thetarget cells, resulting in reverse transcription of the retroviral RNAgenome and integration of the resulting provirus into the cellular DNA.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

II. Overview of Several Embodiments

B. anthracis is the causative agent of anthrax and is surrounded by apolypeptide capsule of poly-γ-D-glutamic acid (γDPGA). γDPGA is poorlyimmunogenic and has antiphagocytic properties. These properties of γDPGAreduce a body's ability to effectively fight B. anthracis infectionusing natural immunity. Thus, the need exists for therapeutic agentsthat prevent, treat, or reduce the effects of B. anthracis infectionand/or to increase B. anthracis clearance from the body. Thus, thisdisclosure provides isolated monoclonal antibodies, chimeric forms ofthese antibodies, humanized forms of these antibodies and fragmentsthereof, that bind the capsule of B. anthracis. Methods of using thedisclosed anti-γDPGA antibodies are also provided.

A Antibodies that Specifically Bind the Capsule of Bacillus anthracis

Disclosed herein are anti-γDPGA monoclonal antibodies that specificallybind poly-γ-D-glutamic acid (γDPGA) on the capsule of B. anthracis, thecausative agent of anthrax, but does not react with cells that do notexpress γDPGA on their surface. In particular embodiments, monoclonalantibodies directed against γDPGA are the anti-γDPGA chimpanzeemonoclonal antibodies 4C and 11D, a consensus anti-γDPGA chimpanzeemonoclonal antibody, chimeric forms thereof, humanized forms thereof,and fragments thereof, wherein any of these antibodies or fragmentsspecifically bind γDPGA on the capsule of B. anthracis.

Generally, the monoclonal antibodies against γDPGA include a variableheavy (V_(H)) and a variable light (V_(L)) chain and specifically bindγDPGA on B. anthracis. The disclosed monoclonal antibodies, includingchimeric forms thereof, humanized forms thereof, or fragments thereof,can specifically bind B. anthracis cells, and can bind the γDPGA antigenof such cells with an affinity constant (Kd) of at least 10⁻⁶ M, such asat least 10⁻⁷ M, at least 10⁻⁸ M, at least 10⁻⁹ M, at least 10⁻¹⁰ M, orat least 10⁻¹¹ M. In specific embodiments, the 4C antibody canspecifically bind B. anthracis cells, and can bind the γDPGA antigen ofsuch cells with an affinity constant of 5.5×10⁻¹⁰ M. In otherembodiments, the 11D antibody can specifically bind B. anthracis cells,and can bind the γDPGA antigen of such cells with an affinity constantof 8.2×10⁻¹¹ M. In yet other embodiments, a consensus anti-γDPGAchimpanzee monoclonal antibody can specifically bind B. anthracis cells,and can bind the γDPGA antigen of such cells with an affinity constantof at least 10⁻⁶ M, such as at least 10⁻⁷ M, at least 10⁻⁸ M, at least10⁻⁹ M, at least 10⁻¹⁰ M, or at least 10⁻¹¹ M.

A consensus anti-γDPGA monoclonal antibody has a consensus light chainamino acid sequence and a consensus heavy chain amino acid sequence,wherein each consensus amino acid sequence represents the results ofmultiple sequence alignments of related sequences and the consensussequence shows amino acid residues that are common among the alignedsequences. Common residues in the consensus light chain and heavy chainsequences are conserved amino acids and are generally important forantigen binding. The residues that vary among the aligned sequencesrepresent amino acid positions that can tolerate sequence variabilityand are generally not important for antigen binding. In some embodimentsof a consensus anti-γDPGA monoclonal antibody, at least one residueposition within the light chain and/or heavy chain consensus amino acidsequences is represented by more than one amino acid. In otherembodiments of a consensus anti-γDPGA monoclonal antibody, at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, or more residuepositions within the light chain and/or heavy chain consensus amino acidsequences is represented by more than one amino acid.

FIG. 1A and FIG. 1B show the amino acid sequence alignments of theanti-γDPGA antibodies 4C and 11D heavy chain and light chain,respectively. The alignments demonstrate the residues in the CDRs thatare conserved between the 4C and 11D monoclonal antibodies. Thisalignment also shows positions that are not conserved between theantibodies, for example the first residue of the V_(H)-CDR1 can beeither a valine (V) or a threonine (T). From this alignment a consensussequence of the heavy chain (SEQ ID NO. 1) and light chain (SEQ ID NO.2) anti-γDPGA antibody is constructed (the consensus anti-γDPGAchimpanzee monoclonal antibody). Thus, disclosed herein is a consensusanti-γDPGA chimpanzee monoclonal antibody. In some embodiments theconsensus anti-γDPGA antibody has a antibody heavy chain variable domainamino acid sequence according to SEQ ID NO: 1. In some embodiments theconsensus anti-γDPGA antibody has a antibody light chain variable domainamino acid sequence according to SEQ ID NO: 2.

In several non-limiting examples, the disclosed monoclonal antibodyincludes at least one of the variable domain light chain and/or at leastone of the variable domain heavy chain amino acid sequences disclosedherein:

Consensus Anti-γDPGA Antibody Heavy Chain Variable Domain Amino AcidSequence:

LEX₁SGGGLVKPGX₂SLX₃LSCAASGFTFSX₄YAMHWVRQAPEKGLEWVSTIGX₅X₆GX₇TX₈X₉SDSVKGRX₁₀X₁₁IX₁₂RDNSX₁₃NTLX₁₄LQMNSLRAEDTAVYYCX₁₅RX₁₆GYCSSTX₁₇CX₁₈SX₁₉X₂₀X₂₁FDX₂₂WGQGTX₂₃VTVS (SEQ ID NO: 1),wherein X₁ can be E or no amino acid; X₂ can be D or G; X₃ can be R orT; X₄ can be V or T; Xs can be A or R; X₆ can be G or S; X₇ can be N orD; X₈ can be W or L; X₉ can be H or Y; X₁₀ can be Y or F; X₁₁ can be Tor S; X₁₂ can be A or S; X₁₃ can be Q or K; X₁₄ can be S or Y; X₁₅ canbe V or A; X₁₆ can be R or K; X₁₇ can be R or N; X₁₈ can be D or Q; X₁₉can be N or Q; X₂₀ can be D or Y; X₂₁ can be A or Y; X₂₂ can be I or Y;or X₂₃ can be M or L.

Consensus Anti-γDPGA Antibody Light Chain Variable Domain Amino AcidSequence:

X₁X₂X₃TQSPSSLSASVGX₄RVX₅ITCRASQDX₆NX₇X₈LAWX₉QQKPGKAPKX₁₀LIX₁₁X₁₂X₁₃SSLQGGVX₁₄SRFSGSGSGTX₁₅FTLTISX₁₆LX₁₇PEDFATYYCX₁₈QX₁₉X₂₀X₂₁YPX₂₂TFGX₂₃GTKX₂₄EIX₂₅RT (SEQ ID NO: 2), wherein X₁ can be A orno amino acid; X₂ can be P or E; X₃ can be M or L; X₄ can be D or G; X₅can be S or T; X₆ can be I or V; X₇ can be D or T; X₈ can be F or W; X₉can be F or Y; X₁₀ can be R or P; X₁₁ can be F or Y; X₁₂ can be R or A;X₁₃ can be T or A; X₁₄ can be S or P; X₁₅ can be E or D; X₁₆ can be N orS; X₁₇ can be R or Q; X₁₈ can be L or Q; X₁₉ can be H or Y; X₂₀ can be Sor K; X₂₁ can be S or H; X₂₂ can be P or L; X₂₃ can be Q or G; X₂₄ canbe L or V or X₂₅ can be S or K.

Exemplary Anti-γDPGA Antibody 4C Heavy Chain Variable Domain Amino AcidSequence:

(SEQ ID NO: 3) LEESGGGLVKPGDSLRLSCAASGFTFSVYAMHWVRQAPEKGLEWVSTIGAGGNTWHSDSVKGRYTIARDNSQNTLSLQMNSLRAEDTAVYYCVRRGYCSSTRCDSNDAFDIWGQGTMVTVS.Exemplary Anti-γDPGA Antibody 4C Light Chain Variable Domain Amino AcidSequence:

(SEQ ID NO: 4) APMTQSPSSLSASVGDRVSITCRASQDINDFLAWFQQKPGKAPKRLIFRTSSLQGGVSSRFSGSGSGTEFTLTISNLRPEDFATYYCLQHSSYPPTFG QGTKLEISRT.Exemplary Anti-γDPGA Antibody 11D Heavy Chain Variable Domain Amino AcidSequence:

(SEQ ID NO: 5) LESGGGLVKPGGSLTLSCAASGFTFSTYAMHWVRQAPEKGLEWVSTIGRSGDTLYSDSVKGRFSISRDNSKNTLYLQMNSLRAEDTAVYYCARKGYCS STNCQSQYYFDYWGQGTLVTVSExemplary Anti-γDPGA Antibody 11D Light Chain Variable Domain Amino AcidSequence:

(SEQ ID NO: 6) ELTQSPSSLSASVGGRVTITCRASQDVNTWLAWYQQKPGKAPKPLIYAASSLQGGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYKHYPLTFGG GTKVEIKRT

In several embodiments, the antibody includes a V_(H) polypeptide havingan amino acid sequence at least about 90% identical, such as at leastabout 95% identical, at least about 98% identical, at least about 99%identical or even 100% identical, to the amino acid sequence set forthas SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 5 and a V_(L) polypeptidehaving an amino acid sequence at least about 90% identical, such as atleast about 95% identical, at least about 98% identical, at least about99% identical or even 100% identical, to the amino acid sequence setforth as SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.

The production of chimeric antibodies (an antibody with non-humanvariable regions and human constant regions) and humanized antibodies(an antibody with non-human SDRs and/or CDRs and human frameworkregions) is well known in the art. Thus chimeric antibodies andhumanized antibodies that specifically bind B. anthracis are disclosed.

In some embodiments, a chimeric antibody that specifically binds γDPGAon B. anthracis is a chimeric form of the 4C or 11D monoclonal antibody,the consensus anti-γDPGA chimpanzee monoclonal antibody, or a functionalfragment thereof. In particular, non-limiting examples of a chimericantibody, the antibody (or antibody fragment) can be an immunoglobulinhaving variable light chain and heavy chain regions from a donormonoclonal antibody (such as 4C or 11D or the consensus anti-γDPGAantibody, see Table 1) that binds B. anthracis, and heavy and lightconstant regions from a human acceptor immunoglobulin. Generally, thechimeric antibody specifically binds to B. anthracis and the γDPGA onsuch cells with an affinity constant of at most 10⁻¹¹ M, such as atleast 10⁻⁶ M, such as at least 10⁻⁷ M, at least 10⁻⁸ M, at least 10⁻⁹ M,at least 10⁻¹⁰ M, or at least 10⁻¹¹ M.

In other embodiments, a humanized antibody that specifically binds B.anthracis is a humanized form of the 4C or 11D monoclonal antibody, theconsensus anti-γDPGA chimpanzee monoclonal antibody, or a functionalfragment thereof. In particular, non-limiting examples of the humanizedantibody, the antibody (or antibody fragment) can be an immunoglobulinhaving complementarity determining regions (CDRs) from a donormonoclonal antibody (such as 4C or 11D or the consensus anti-γDPGAantibody, see Table 1) that binds B. anthracis, and heavy chain andlight chain variable region frameworks from the same or different humanacceptor immunoglobulins. Generally, the humanized antibody specificallybinds to B. anthracis with an affinity constant of at most 10⁻¹¹ M, suchas at least 10⁻⁶ M, such as at least 10⁻⁷ M, at least 10⁻⁸ M, at least10⁻⁹ M, at least 10⁻¹⁰ M, or at least 10⁻¹¹ M. The location of the CDRswithin the variable light chain and variable heavy chain amino acidsequences for the consensus anti-γDPGA chimpanzee monoclonal antibody,and for the 4C and 11D antibodies, are set forth in Table 1 below:

TABLE 1 Antibody chain Sequence Consensus V_(H)-CDR1 Amino acid 28-32 ofSEQ ID NO: 1 Consensus V_(H)-CDR2 Amino acid 47-55 of SEQ ID NO: 1Consensus V_(H)-CDR3 Amino acid 95-111 of SEQ ID NO: 1 ConsensusV_(L)-CDR1 Amino acid 23-33 of SEQ ID NO: 2 Consensus V_(L)-CDR2 Aminoacid 48-55 of SEQ ID NO: 2 Consensus V_(L)-CDR3 Amino acid 88-96 of SEQID NO: 2  4C V_(H)-CDR1 Amino acid 28-32 of SEQ ID NO: 3  4C V_(H)-CDR2Amino acid 47-55 of SEQ ID NO: 3  4C V_(H)-CDR3 Amino acid 95-111 of SEQID NO: 3  4C V_(L)-CDR1 Amino acid 23-33 of SEQ ID NO: 4  4C V_(L)-CDR2Amino acid 48-55 of SEQ ID NO: 4  4C V_(L)-CDR3 Amino acid 88-96 of SEQID NO: 4 11D V_(H)-CDR1 Amino acid 27-31 of SEQ ID NO: 5 11D V_(H)-CDR2Amino acid 46-54 of SEQ ID NO: 5 11D V_(H)-CDR3 Amino acid 94-11- of SEQID NO: 5 11D V_(L)-CDR1 Amino acid 22-32 of SEQ ID NO: 6 11D V_(L)-CDR2Amino acid 47-54 of SEQ ID NO: 6 11D V_(L)-CDR3 Amino acid 87 to 95 ofSEQ ID NO: 6

In some embodiments, a humanized antibody includes one or more CDRs fromthe variable region of the heavy chain of the consensus anti-γDPGAchimpanzee monoclonal antibody. Thus in some embodiments a humanizedantibody includes amino acids 28-32 of SEQ ID NO: 1 (HCDR1), amino acids47-55 of SEQ ID NO: 1 (HCDR2), or amino acids 95-111 of SEQ ID NO: 1(HCDR3), wherein X₄ is V or T; X₅ is A or R; X₆ is G or S; X₇ is N or D;X₈ is W or L; X₁₆ is R or K; X₁₇ is R or N; X₁₈ is D or Q; X₁₀ is N orQ; X₂₀ is D or Y; X₂₁ is A or Y; or X₂₂ is I or Y. The antibodyspecifically binds γDPGA.

In other embodiments, a humanized antibody includes the CDRs from thevariable region of the light chain of the consensus anti-γDPGAchimpanzee monoclonal antibody. Thus in some embodiments a humanizedantibody includes amino acids 23-33 of SEQ ID NO: 2 (LCDR1), amino acids48-55 of SEQ ID NO: 2 (LCDR2), or amino acids 88-96 of SEQ ID NO: 2(LCDR3), wherein X₄ is V or T; X₅ is A or R; X₆ is G or S; X₇ is N or D;X₈ is W or L; X₁₆ is R or K; X₁₇ is R or N; X₁₈ is D or Q; X₁₉ is N orQ; X₂₀ is D or Y; X₂₁ is A or Y; and X₂₂ is I or Y; wherein X₆ is I orV; X₇ is D or T; X₈ is F or W; X₁₁ is F or Y; X₁₂ is R or A; X₁₃ is T orA; X₁₈ is L or Q; X₁₉ is H or Y; X₂₀ is S or K; X₂₁ is S or H; or X₂₂ isP or L.

In particular embodiments, a humanized antibody includes the CDRs fromthe variable region of the heavy chain of the chimpanzee monoclonalantibody 4C. Thus, in some embodiments, a humanized antibody includesamino acids 28-32 of SEQ ID NO: 3 (HCDR1), amino acids 47-55 of SEQ IDNO: 3 (HCDR2), or amino acids 95-111 of SEQ ID NO: 3 (HCDR3). In certainembodiments, a humanized antibody includes the CDRs from the variableregion of the light chain of the chimpanzee monoclonal antibody 4C.Thus, in some embodiments, a humanized antibody includes amino acids23-33 of SEQ ID NO: 4 (LCDR1), amino acids 48-55 of SEQ ID NO: 4(LCDR2), or amino acids 88-96 of SEQ ID NO: 4 (LCDR3).

In some embodiments, a humanized antibody includes the CDRs from thevariable region of the heavy chain of the chimpanzee monoclonal antibody11D. Thus, in some embodiments, a chimeric antibody includes amino acids27-31 of SEQ ID NO: 5 (HCDR1), amino acids 46-54 of SEQ ID NO: 5(HCDR2), or amino acids 94-110 of SEQ ID NO: 5 (HCDR3). In otherembodiments, a humanized antibody includes the CDRs from the variableregion of the light chain of the chimpanzee monoclonal antibody 11D.Thus, in particular embodiments, a humanized antibody includes aminoacids 22-32 of SEQ ID NO: 6 (LCDR1), amino acids 47-54 of SEQ ID NO: 6(LCDR2), or amino acids 87-95 of SEQ ID NO: 6 (LCDR3).

Humanized monoclonal antibodies can be produced by transferring donorCDRs from heavy and light variable chains of the donor chimpanzeeimmunoglobulin (such as the consensus anti-γDPGA chimpanzee monoclonalantibody, the chimpanzee monoclonal antibody 4C, or the chimpanzeemonoclonal antibody 11D) into a human framework region. In someembodiments, human residues in the framework regions are substitutedwith residues from the donor antibody, if required to retain affinity.Techniques for producing humanized monoclonal antibodies are described,for example, by Jones et al., Nature 321:522, 1986; Riechmann et al.,Nature 332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carteret al., Proc. Nat'l Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev.Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993.The antibody may be of any isotype, for example, IgA, IgD, IgE, IgG, orIgM. Specific non-limiting examples of subclass of an IgG antibodyinclude IgG1, IgG2, IgG3, or IgG4.

In one embodiment, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 65% identical tothe sequence of the donor immunoglobulin heavy chain variable regionframework. In other embodiments, the sequence of the humanizedimmunoglobulin heavy chain variable region framework can be at leastabout 75%, at least about 85%, at least about 95%, at least about 98%,or at least about 99% identical to the sequence of the donorimmunoglobulin heavy chain variable region framework. The sequences ofthe heavy and light chain frameworks are known in the art. Humanframework regions, and mutations that can be made in a humanizedantibody framework regions, are known in the art (see, for example, inU.S. Pat. No. 5,585,089, which is incorporated herein by reference).

Exemplary human antibodies LEN and 21/28′CL are of use in providingframework regions. Exemplary light chain frameworks of human monoclonalantibody LEN have the following sequences:

(SEQ ID NO: 11) FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 12)FR2: WYQQKPGQPPLLIY (SEQ ID NO: 13)FR3: GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 14) FR4: FGQGQTKLEIK

Exemplary heavy chain frameworks of human monoclonal antibody 21/28′CLhave the following sequences:

(SEQ ID NO: 15) FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ ID NO: 16)FR2: WVRQAPGQRLEWMG (SEQ ID NO: 17)FR3: RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 18) FR4: WGQGTLVTVSS.

The disclosed antibodies can be of any isotype. The monoclonal antibodycan be, for example, an IgM or an IgG antibody, such as IgG1 or an IgG2.In one embodiment, a nucleic acid molecule encoding V_(L) or V_(H) isisolated using methods well-known in the art, such that it does notinclude any nucleic acid sequences encoding the constant region of thelight or heavy chain, respectively. The nucleic acid molecule encodingV_(L) or V_(H) is then operatively linked to a nucleic acid sequenceencoding a light chain constant region (C_(L)) or heavy chain constantregion (C_(H)) from a different isotype or class of immunoglobulinmolecule. This can be achieved using a vector or nucleic acid moleculethat comprises a C_(L) or C_(H) chain. Nucleic acid molecules encodingsuch sequences are known in the art. In some examples, the regions of anantibody that determine the antibody isotype or class can be substitutedwith the corresponding region of another antibody isotype or class usingrecombinant methods. For example, an antibody that specifically binds B.anthracis that is an IgM may be switched to an IgG. In particular,non-limiting examples, class switching can be used to convert one IgGsubclass to another, such as from IgG1 to IgG2, IgG3, or IgG4. In otherembodiments, an IgG constant region can be added to an antibody fragmenthaving the antigen binding domains, such as a Fab fragment. In aspecific non-limiting example, a constant region from a human IgG1(human γ1 constant region) is combined with a chimpanzee Fab region.

Antibody fragments, such as Fab, F(ab)₂, and Fv which include a heavychain and light chain variable region and are capable of binding theepitopic determinant on B. anthracis, are encompassed by the presentdisclosure. These antibody fragments retain the ability to selectivelybind with the antigen. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV2), defined as a dimer of anscFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). Antibodies, such as chimpanzee monoclonalantibodies, chimeric antibodies, and humanized antibodies, include fulllength molecules as well as fragments thereof, such as Fab, F(ab′)₂, andFv which include a heavy chain and light chain variable region and arecapable of binding the epitopic determinant. In some embodiments, theantibodies have V_(L) (for example, SEQ ID NO: 2, SEQ ID NO: 4; or SEQID NO: 6) and V_(H) (for example, SEQ ID NO: 1, SEQ ID NO: 3; or SEQ IDNO: 5) regions, or portions thereof, such as the CDRs or SDRs. Fvantibodies are typically about 25 kDa and contain a completeantigen-binding site with three CDRs per each heavy chain and each lightchain. To produce these antibodies, the V_(H) and the V_(L) can beexpressed from two individual nucleic acid constructs in a host cell. Ifthe V_(H) and the V_(L) are expressed non-contiguously, the chains ofthe Fv antibody are typically held together by noncovalent interactions.However, these chains tend to dissociate upon dilution, so methods havebeen developed to crosslink the chains through glutaraldehyde,intermolecular disulfides, or a peptide linker. Thus, in one example,the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chainvariable region and the light chain variable region are chemicallylinked by disulfide bonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (sFv) are prepared by constructing a structural gene comprisingDNA sequences encoding the V_(H) and V_(L) domains connected by anoligonucleotide. The structural gene is inserted into an expressionvector, which is subsequently introduced into a host cell such as E.coli. The recombinant host cells synthesize a single polypeptide chainwith a linker peptide bridging the two V domains. Methods for producingsFvs are known in the art (see Whitlow et al., Methods: a Companion toMethods in Enzymology, Vol. 2, page 97, 1991; Bird et al., Science242:423, 1988; U.S. Pat. No. 4,946,778; and Pack et al., Bio/Technology11:1271, 1993).

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review SEQ ID NOs: 1-6 locateone or more of the amino acids in the brief table above, identify aconservative substitution, and produce the conservative variant usingwell-known molecular biology techniques. Generally, conservativevariants will bind the target antigen with an equal to or greaterefficiency than the parent monoclonal antibody.

Monoclonal antibodies may be produced to either the normal γDPGA proteinor mutant forms of this protein. In one embodiment, monoclonalantibodies to γDPGA can be prepared by hybridoma fusion. A monoclonalantibody to epitopes of γDPGA, identified and isolated as described, canbe prepared from murine hybridomas according to the classical method ofKohler and Milstein (Nature 256:495-497, 1975) or derivative methodsthereof. In one specific, non-limiting embodiment, a mouse isrepetitively inoculated with a few micrograms of the selected proteinover a period of a few weeks. The mouse is then sacrificed, and theantibody-producing cells of the spleen isolated. The spleen cells arefused with mouse myeloma cells using polyethylene glycol, and theexcess, non-fused, cells destroyed by growth of the system on selectivemedia comprising aminopterin (HAT media). Successfully fused cells arediluted and aliquots of the dilution placed in wells of a microtiterplate, where growth of the culture is continued. Antibody-producingclones are identified by detection of antibody in the supernatant fluidof the wells by immunoassay procedures, such as ELISA, as originallydescribed by Engvall (Enzymol. 70(A):419-439, 1980), and derivativemethods thereof. Selected positive clones can be expanded and theirmonoclonal antibody product harvested for use. Detailed procedures formonoclonal antibody production are described in Harlow and Lane(Antibodies, A Laboratory Manual, CSHL, New York, 1988). Hybridomacells, and their progeny, that secrete monoclonal antibodies are alsoencompassed by this disclosure.

In another embodiment, recombinant methods are used to preparemonoclonal antibodies against γDPGA. In one specific, non-limitingexample to obtain therapeutically useful anti-γDPGA monoclonalantibodies, a host, such as a chimpanzee, can be immunized withconjugates of a 10 or 15-mer of D-glutamic acid polymers (γDPGA) boundto an immunogenic carrier protein such as recombinant protective antigen(rPA) or tetanus toxoid (TT). After several immunizations, the hostdevelops strong immune responses to γDPGA. A combinatorial Fab libraryof mRNA derived from the host's bone marrow can be prepared and distinctclones expressing Fabs reactive with native γDPGA recovered. Fabs can beconverted into full-length IgG with human 71 heavy chain constantregions. Such monoclonal antibodies can be tested for opsonophagocytickilling of bacilli in an in vitro assay. As chimpanzee immunoglobulinsare virtually identical to human immunoglobulins, these chimpanzeeanti-γDPGA monoclonal antibodies have clinically useful applications.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody that specifically binds γDPGA onB. anthracis, using any number of means known to those of skill in theart. Both covalent and noncovalent attachment means may be used. Theprocedure for attaching an effector molecule to an antibody variesaccording to the chemical structure of the effector. Polypeptidestypically contain a variety of functional groups (such as carboxylicacid (COOH), free amine (—NH₂) or sulfhydryl (—SH) groups) which areavailable for reaction with a suitable functional group on an antibodyto result in the binding of the effector molecule. Alternatively, theantibody is derivatized to expose or attach additional reactivefunctional groups. The derivatization may involve attachment of any of anumber of linker molecules such as those available from Pierce ChemicalCompany (Rockford, Ill.). The linker can be any molecule used to jointhe antibody to the effector molecule. The linker is capable of formingcovalent bonds to both the antibody and to the effector molecule.Suitable linkers are well known to those of skill in the art andinclude, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. Where theantibody and the effector molecule are polypeptides, the linkers may bejoined to the constituent amino acids through their side groups (such asthrough a disulfide linkage to cysteine) or to the alpha carbon aminoand carboxyl groups of the terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (forexample when exposed to tumor-associated enzymes or acidic pH) may beused.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules), and otheragents to antibodies, one skilled in the art will be able to determine asuitable method for attaching a given agent to an antibody or otherpolypeptide. In some examples a label is a detectable label such as afluorophore (for example FTIC, PE and the like), an enzyme (for exampleHRP), a radiolabel, or a nanoparticle (for example a gold particle or asemiconductor nanocrystal, such as a quantum dot (QDOT®)).

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibodies or portion thereof is derivatized such that thebinding to γDPGA on B. anthracis is not affected adversely by thederivatization or labeling. For example, the antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate associate of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, such as to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company (Rockford, Ill.).

An antibody that specifically binds γDPGA on B. anthracis can be labeledwith a detectable moiety. Useful detection agents include fluorescentcompounds, including fluorescein, fluorescein isothiocyanate, rhodamine,5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanidephosphors and the like. Bioluminescent markers are also of use, such asluciferase, Green fluorescent protein (GFP), Yellow fluorescent protein(YFP). An antibody can also be labeled with enzymes that are useful fordetection, such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, glucose oxidase and the like. When an antibody islabeled with a detectable enzyme, it can be detected by addingadditional reagents that the enzyme uses to produce a reaction productthat can be discerned. For example, when the agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Itshould be noted that the avidin itself can be labeled with an enzyme ora fluorescent label.

An antibody may be labeled with a magnetic agent, such as gadolinium.Antibodies can also be labeled with lanthanides (such as europium anddysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce potential steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect Bacillus anthracis byx-ray, emission spectra, or other diagnostic techniques. Examples oflabels for polypeptides include, but are not limited to, the followingradioisotopes or radionuclides: ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In,¹²⁵I, ¹³¹I.

Nucleic acids encoding the amino acid sequences of the antibodies thatbind γDPGA on B. anthracis are also provided herein. Exemplary nucleicacid sequences are as follows:

Anti-γDPGA 4C Heavy Chain Variable Domain DNA Sequence:

(SEQ ID: 7)ctcgaggagtctgggggaggcctggtaaagcctggggattccctgagactctcgtgtgcagcctctggattcaccttcagtgtctatgctatgcactgggtccgccaggctccagagaaggggctggagtgggtctcaactattggtgctggtggtaatacgtggcactccgactctgtcaagggccgatacaccattgccagagacaattcccagaatacgctgtctctgcaaatgaacagcctgagagccgaggacacggccgtgtattactgtgtgagaaggggatactgtagcagtactaggtgcgacagtaatgatgcttttgatatctggggccaagggacaatggtcaccgtctctAnti-γDPGA 4C Light Chain Variable Domain DNA Sequence:

(SEQ ID: 8)gctccgatgacccagtctccatcctcattgtctgcatctgtgggagacagagtcagcatcacttgtcgggcgagtcaggacattaacgattattggcctggatcagcagaaaccagggaaagcccctaagcgtctgatattcgtacttccagtagcaaggtggagtctcatcaagattcagtggcagtggatctgggacagaattcactctcacaatcagcaacctgcggcctgaagattttgcaacttattactgtctgcagcatagacttaccctccgaccacggccaagggaccaaactggagatcagccgaactAnti-γDPGA 11D Heavy Chain Variable Domain DNA Sequence:

(SEQ ID: 9)ctcgagtctgggggaggcaggtcaagccgggggggtccctgacactctcgtgtgcagcctctggattcaccacagtacctatgctatgcactgggtccgccaggctccagagaaggggctggagtgggtctcaactattggtcgtagtggtgacacgttgtactcagactctgtcaagggccgattcagcatctccagagacaattccaagaacaccctgtatctgcaaatgaacagcctgagagccgaggacacggccgtgtattattgtgcgagaaagggatattgtagtagtaccaactgtcagtcccaatattactttgactactggggccagggaaccctggtcaccgtctccAnti-γDPGA 11D Light Chain Variable Domain DNA Sequence:

(SEQ ID: 10)gagctcacccagtctccatcctcactgtctgcatctgtgggaggcagagtcaccatcacttgtcgggccagtcaggatgttaacacctggttagcctggtatcagcagaaaccagggaaagcccctaagcccctgatctatgctgcatccagtagcaaggtggggtcccatcaaggatagcggcagtgggtctgggacagatttcactctaaccatcagcagcctgcagcctgaagattttgcaacttattactgccaacaatataaacattaccctctcactacggtggagggaccaaggtggagatcaaacgaact

Nucleotides molecules encoding the antibodies can readily be produced byone of skill in the art, using the amino acid sequences provided herein,and the genetic code. In addition, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which differ in sequence but which encode one antibodysequence. Thus, nucleic acids encoding antibodies, conjugates and fusionproteins are provided herein. Nucleic acid molecules encoding the 4C and11D Fab sequences were deposited with the ATCC on Nov. 14, 2008, inaccordance with the Budapest Treaty (Accession Nos. PTA-9610 andPTA-9609, respectively).

Nucleic acid sequences encoding the human antibodies that specificallybind γDPGA on B. anthracis can be prepared by any suitable methodincluding, for example, cloning of appropriate sequences or by directchemical synthesis by methods such as the phosphotriester method ofNarang et al., Meth. Enzymol. 68:90-99, 1979; the phosphodiester methodof Brown et al., Meth. Enzymol. 68:109-151, 1979; thediethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill would recognize that while chemicalsynthesis of DNA is generally limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding a human antibodythat specifically binds γDPGA on B. anthracis can be prepared by cloningtechniques. Examples of appropriate cloning and sequencing techniques,and instructions sufficient to direct persons of skill through manycloning exercises are found in Sambrook et al., supra, Berger and Kimmel(eds.), supra, and Ausubel, supra. Product information frommanufacturers of biological reagents and experimental equipment alsoprovide useful information. Such manufacturers include the SIGMAChemical Company (Saint Louis, Mo.), R&D Systems (Minneapolis, Minn.),Pharmacia Amersham (Piscataway, N.J.), CLONTECH Laboratories, Inc. (PaloAlto, Calif.), Chem Genes Corp., Aldrich Chemical Company (Milwaukee,Wis.), Glen Research, Inc., GIBCO BRL Life Technologies, Inc.(Gaithersburg, Md.), Fluka Chemica-Biochemika Analytika (Fluka ChemieAG, Buchs, Switzerland), Invitrogen (San Diego, Calif.), and AppliedBiosystems (Foster City, Calif.), as well as many other commercialsources known to one of skill in the art.

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill.

In one example, an antibody of use is prepared by inserting the cDNAwhich encodes a variable region from an antibody into a vector whichcomprises the cDNA encoding an effector molecule (EM), such as an enzymeor label. The insertion is made so that the variable region and the EMare read in frame so that one continuous polypeptide is produced. Thus,the encoded polypeptide contains a functional Fv region and a functionalEM region. In one embodiment, cDNA encoding an enzyme is ligated to ascFv so that the enzyme is located at the carboxyl terminus of the scFv.In several examples, cDNA encoding a horseradish peroxidase or alkalinephosphatase, or a polypeptide marker of interest is ligated to a scFv sothat the enzyme (or polypeptide marker) is located at the amino terminusof the scFv. In another example, the label is located at the aminoterminus of the scFv. In a further example, cDNA encoding the protein orpolypeptide marker is ligated to a heavy chain variable region of anantibody, so that the enzyme or polypeptide marker is located at thecarboxyl terminus of the heavy chain variable region. The heavychain-variable region can subsequently be ligated to a light chainvariable region of the antibody using disulfide bonds. In a yet anotherexample, cDNA encoding an enzyme or a polypeptide marker is ligated to alight chain variable region of an antibody, so that the enzyme orpolypeptide marker is located at the carboxyl terminus of the lightchain variable region. The light chain-variable region can subsequentlybe ligated to a heavy chain variable region of the antibody usingdisulfide bonds.

Once the nucleic acids encoding the antibody, labeled antibody, orfragment thereof are isolated and cloned, the protein can be expressedin a recombinantly engineered cell such as bacteria, plant, yeast,insect and mammalian cells using a suitable expression vector. One ormore DNA sequences encoding the antibody or fragment thereof can beexpressed in vitro by DNA transfer into a suitable host cell. The cellmay be prokaryotic or eukaryotic. The term also includes any progeny ofthe subject host cell. It is understood that all progeny may not beidentical to the parental cell since there may be mutations that occurduring replication. Methods of stable transfer, meaning that the foreignDNA is continuously maintained in the host, are known in the art.Hybridomas expressing the antibodies of interest are also encompassed bythis disclosure.

Polynucleotide sequences encoding the antibody, labeled antibody, orfunctional fragment thereof, can be operatively linked to expressioncontrol sequences. An expression control sequence operatively linked toa coding sequence is ligated such that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody, labeled antibody, orfunctional fragment thereof can be inserted into an expression vectorincluding, but not limited to, a plasmid, virus or other vehicle thatcan be manipulated to allow insertion or incorporation of sequences andcan be expressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect, and mammalian organisms. Methods of expressingDNA sequences having eukaryotic or viral sequences in prokaryotes arewell known in the art. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart. Nucleic acid molecules encoding the 4C and 11D Fab sequences weredeposited with the ATCC on Nov. 14, 2008, in accordance with theBudapest Treaty (Accession Nos. PTA-9610 and PTA-9609, respectively). Inone specific, non-limiting example, an expression vector encoding the 4CFab sequence is pComb3H-4C, deposited with the ATCC on Nov. 14, 2008, inaccordance with the Budapest Treaty (Accession No. PTA-9610). In anotherspecific, non-limiting example, an expression vector encoding the 11DFab sequence is pComb3H-11D, deposited with the ATCC on Nov. 14, 2008,in accordance with the Budapest Treaty (Accession No. PTA-9609).

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques, as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbCl canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, methods of DNA transfection, such ascalcium phosphate co-precipitation, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the disclosedantibody, labeled antibody, or functional fragment thereof, and a secondforeign DNA molecule encoding a selectable phenotype, such as the herpessimplex thymidine kinase gene. Another method is to use a eukaryoticviral vector, such as simian virus 40 (SV40) or bovine papilloma virus,to transiently infect or transform eukaryotic cells and express theprotein (see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the antibody, labeledantibody or functional fragment thereof can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.,1982). Substantially pure compositions of at least about 90% to 95%, 95%to 98%, or 98% to 99% homogeneity are disclosed herein, and 98 to 99% ormore homogeneity can be used for pharmaceutical purposes. Once purified,partially or to homogeneity as desired, if to be used therapeutically,the polypeptides should be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

B Therapeutic Compositions

Compositions, such as therapeutic or pharmaceutical compositions, areprovided that include one or more of the disclosed anti-γDPGAantibodies, humanized forms thereof, chimeric forms thereof, orfragments thereof that specifically bind B. anthracis. The antibodies orfragments thereof can be administered in vitro, ex vivo, or in vivo to acell or subject. It is desirable to prepare the antibodies or fragmentsthereof as a pharmaceutical composition appropriate for the intendedapplication, for example to inhibit or treat a B. anthracis infection orfor the detection of a B. anthracis infection. Accordingly, methods formaking a medicament or pharmaceutical composition containing a disclosedanti-γDPGA antibody or fragment thereof, alone or in combination withother agents that inhibit, treat, or detect B. anthracis infection, aredisclosed herein. In some embodiments, the disclosed anti-γDPGAantibodies or fragments thereof are administered with one or moreisolated anti-B. anthracis (anti-anthrax) antibodies or fragmentthereof, such as anti-anthrax toxin antibodies. In particularembodiments, anti-anthrax toxin antibodies include one or more of ananti-PA, -LF or -EF antibody or fragment thereof (for example, theanti-PA, -LF and -EF antibodies disclosed in International patentpublication Nos. WO08103845 and WO07084107, both of which areincorporated herein by reference in their entirety) are included herein.Anti-γDPGA antibodies can be prepared for administration alone or withother agents, such as antibiotics (for example one or more isolated antianthrax toxin antibodies or fragment thereof, such as one or more of ananti-PA, -LF or -EF antibody or fragment thereof) and/or other proteins,such as with complement protein or fragments thereof.

In some examples, a therapeutic composition includes a disclosedanti-γDPGA antibody. In other examples, a therapeutic compositionincludes one or more isolated anti-anthrax toxin antibodies or fragmentthereof, such as one or more of an anti-PA, -LF and -EF antibody orfragment thereof. In yet other examples, a therapeutic compositionincludes a disclosed anti-DPGA antibody or a fragment thereof and one ormore isolated anti anthrax toxin antibodies or a fragment thereof, suchas one or more of an anti-PA, -LF and -EF antibody.

Typically, preparation of a pharmaceutical composition (for use as amedicament or in the manufacture of a medicament) entails preparing apharmaceutical composition that is essentially free of pyrogens, as wellas any other impurities that could be harmful to humans or animals.Typically, the pharmaceutical composition contains appropriate salts andbuffers to render the components of the composition stable and allow thedisclosed anti-γDPGA antibody or fragment thereof to interact with B.anthracis cells that are in a subject.

Antibodies may be provided in lyophilized form and rehydrated withsterile water before administration, although they are also provided insterile solutions of known concentration. In some examples, the antibodysolution is then added to an infusion bag containing 0.9% sodiumchloride, USP, and typically administered at a dosage of from 0.5 to 15mg/kg of body weight. Considerable experience is available in the art inthe administration of antibody drugs, which have been marketed in theU.S. since the approval of RITUXAN® in 1997. Antibodies can beadministered by slow infusion, rather than in an intravenous push orbolus. In one example, a higher loading dose is administered, withsubsequent, maintenance doses being administered at a lower level. Forexample, an initial loading dose of 4 mg/kg may be infused over a periodof some 90 minutes, followed by weekly maintenance doses for 4-8 weeksof 2 mg/kg infused over a 30 minute period if the previous dose was welltolerated.

Therapeutic compositions can be provided as parenteral compositions,such as for injection or infusion. Such compositions are formulatedgenerally by mixing a disclosed anti-γDPGA antibody or fragment thereofand/or one or more isolated anti anthrax toxin antibodies or fragmentthereof, such as one or more of an anti-PA, -LF and -EF antibody orfragment thereof, at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, for example one that is non-toxicto recipients at the dosages and concentrations employed and iscompatible with other ingredients of the formulation. In addition, adisclosed anti-γDPGA antibody or fragment thereof and/or one or moreisolated anti anthrax toxin antibodies or fragment thereof, such as oneor more of an anti-PA, -LF and -EF antibody or fragment thereof, can besuspended in an aqueous carrier, for example, in an isotonic buffersolution at a pH of about 3.0 to about 8.0, preferably at a pH of about3.5 to about 7.4, 3.5 to 6.0, or 3.5 to about 5.0. Useful buffersinclude sodium citrate-citric acid and sodium phosphate-phosphoric acid,and sodium acetate/acetic acid buffers. The disclosed anti-γDPGAantibody or fragment thereof, optionally together with excipients and/orone or more isolated anti anthrax toxin antibodies or fragment thereof,such as one or more of an anti-PA, -LF and -EF antibody or fragmentthereof, can also be in the form of a lyophilisate and can be made intoa solution prior to parenteral administration by the addition ofsuitable solvents. Solutions such as those that are used, for example,for parenteral administration can also be used as infusion solutions.

Pharmaceutical compositions can include an effective amount (such as atherapeutically effective amount) of a disclosed anti-γDPGA antibody orfragment thereof, and/or one or more isolated anti-anthrax toxinantibodies or fragment thereof, such as one or more of an anti-PA, -LFor -EF antibody or fragment thereof in a pharmaceutically acceptablecarrier or excipient. Pharmaceutically acceptable carriers and/orpharmaceutically acceptable excipients are known in the art and aredescribed, for example, in Remington's Pharmaceutical Sciences, by E. W.Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975).

The nature of the carrier will depend on the particular mode ofadministration being employed. For example, parenteral formulationsusually contain injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (such as powder, pill, tablet, orcapsule forms), conventional non-toxic solid carriers can include, forexample, pharmaceutical grades of mannitol, lactose, starch or magnesiumstearate. In addition, pharmaceutical compositions to be administeredcan contain minor amounts of non-toxic auxiliary substances, such aswetting or emulsifying agents, preservatives, and pH buffering agentsand the like, for example sodium acetate or sorbitan monolaurate.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions. For example, certainpharmaceutical compositions can include a disclosed anti-γDPGA antibodyor fragment thereof and/or one or more isolated anti-anthrax toxinantibodies or fragment thereof, such as one or more of an anti-PA, -LFor -EF antibody or fragment thereof in water, mixed with a suitablesurfactant, such as hydroxypropylcellulose. Dispersions also can beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

Additional formulations are suitable for oral administration. Oralformulations can include excipients such as, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like. The compositions(medicaments) typically take the form of solutions, suspensions,aerosols or powders. Exemplary formulations can be found in U.S. Patentpublication No. 20020031527. When the route is topical, the form may bea cream, ointment, salve or spray.

Typical subjects intended for treatment with the pharmaceuticalcompositions and methods of the present disclosure include humans, aswell as non-human primates and other animals. To identify subjects forprophylaxis or treatment according to the methods of the disclosure,accepted screening methods are employed to determine risk factorsassociated with a targeted or suspected disease or condition (forexample, an infection associated with a particular pathogen of interest,such as B. anthracis) or to determine the status of an existing diseaseor condition in a subject. These screening methods include, for example,diagnostic methods, such as various ELISA and other immunoassay methods,which are available and well known in the art to detect and/orcharacterize disease-associated markers. These and other routine methodsallow the clinician to select patients in need of therapy using themethods and pharmaceutical compositions of the disclosure.

C. Methods of Treatment

Methods of treating or inhibiting a B. anthracis infection in a subjectare disclosed. Subjects that would benefit from such methods include forexample, a subject that has a B. anthracis infection or is at risk ofdeveloping a B. anthracis infection, such as a subject that has beenexposed or is believed to have been exposed to B. anthracis, for examplea subject exposed or potentially exposed to B. anthracis spores. Inparticular examples, the method is used to enhance theopsonophagocytosis of B. anthracis in a subject who is infected with (orat risk of being infected with) B. anthracis. In some examples a subjectis selected for treatment that has or is at risk for developing aninfection by B. anthracis, for example a prophylactic administration.The methods include administering to the subject a therapeuticallyeffective amount of one or more of the anti-γDPGA antibodies, orfragments thereof disclosed herein. In some examples, a subject isadministered one or more of the 4C monoclonal antibody, the 11Dmonoclonal antibody, the consensus anti-γDPGA chimpanzee monoclonalantibody, a chimeric form thereof, or a humanized form thereof, or afragment thereof.

Without being bound by theory, the anti-γDPGA monoclonal antibodies 4Cand 11D can function as opsonophagocytotic antibodies. Thus, in someembodiments, the administration of the 4C monoclonal antibody, the 11Dmonoclonal antibody, the consensus anti-γDPGA chimpanzee monoclonalantibody, a chimeric form thereof, or a humanized form thereof, or afragment thereof enhances the ability of the subject's immune system(and specifically the ability of effector cells, such as macrophages,eosinophils, and neutrophils, of the subject) to opsonophagocytose B.anthracis as a result of the B. anthracis being specifically bound bythe 4C monoclonal antibody, the 11D monoclonal antibody, the consensusanti-γDPGA chimpanzee monoclonal antibody, a chimeric form thereof, or ahumanized form thereof, or a fragment thereof. Complement proteins andfragments thereof assist in opsonophagocytic killing of pathogens bybinding to opsonic antibodies, such as the 4C monoclonal antibody or the11D monoclonal antibody, and facilitating the opsonization by effectorcells.

It is contemplated that the disclosed anti-γDPGA antibodies can work inconjunction with anti-anthrax toxin antibodies or fragment thereof toinhibit and/or treat infection by B. anthracis. Thus, in some examples,the subject is also administered a pharmaceutically effective amount ofone or more isolated anti-anthrax toxin antibodies or fragment thereof,such as one or more of an anti-PA, -LF or -EF antibody or fragmentthereof. Examples of anti-anthrax toxin antibodies or fragment thereof,such as anti-PA, -LF or -EF antibodies, can be found in Internationalpatent publication Nos. WO08103845 and WO07084107, both of which areincorporated herein by reference in their entirety. In some examples thedisclosed anti-γDPGA antibodies are administered concurrently with oneor more of isolated anti-anthrax toxin antibodies or fragment thereof,such as one or more of an anti-PA, -LF or -EF antibody or fragmentthereof. In some example the disclosed anti-γDPGA antibodies areadministered sequentially with one or more of isolated anti-anthraxtoxin antibodies or fragment thereof, such as one or more of an anti-PA,-LF or -EF antibody or fragment thereof. Sequential administration canbe separated by any amount of time so long as the desired effect areachieved, for example prevention or treatment of an anthrax infection.Multiple administrations of the compositions described herein are alsocontemplated.

Administration of therapeutic compositions can be by any common route aslong as the target tissue is available via that route. This includesoral, nasal (such as intranasal), ocular, buccal, enteral, intravitral,or other mucosal (such as rectal or vaginal) or topical administration.Alternatively, administration will be by orthotopic, intradermalsubcutaneous, intramuscular, parentral intraperitoneal, or intravenousinjection routes. Such pharmaceutical compositions are usuallyadministered as pharmaceutically acceptable compositions that includephysiologically acceptable carriers, buffers or other excipients.

An effective amount of the pharmaceutical composition is determinedbased on the intended goal, for example to inhibit and/or treat apathogenic infection of a human or non-human subject. The administrationof the pharmaceutical compositions of the disclosure can be for eitherprophylactic or therapeutic purpose. When provided prophylactically, thepharmaceutical composition is provided in advance of any symptom. Theprophylactic administration of the compound serves to prevent orameliorate any subsequent disease process. When providedtherapeutically, the compound is provided at (or shortly after) theonset of a symptom of disease or infection.

For prophylactic and therapeutic purposes, the pharmaceuticalcompositions can be administered to the subject in a single bolusdelivery, via continuous delivery (for example, continuous transdermal,mucosal or intravenous delivery) over an extended time period, or in arepeated administration protocol (for example, by an hourly, daily orweekly, repeated administration protocol). The therapeutically effectivedosage of the compound can be provided as repeated doses within aprolonged prophylaxis or treatment regimen that will yield clinicallysignificant results to alleviate one or more symptoms or detectableconditions associated with a targeted disease or condition as set forthherein. Determination of effective dosages in this context is typicallybased on animal model studies followed up by human clinical trials andis guided by administration protocols that significantly reduce theoccurrence or severity of targeted disease symptoms or conditions in thesubject. Suitable models in this regard include, for example, murine,rat, porcine, feline, non-human primate, and other accepted animal modelsubjects known in the art. Alternatively, effective dosages can bedetermined using in vitro models (for example, immunologic andhistopathologic assays). Using such models, only ordinary calculationsand adjustments are required to determine an appropriate concentrationand dose to administer a therapeutically effective amount of a disclosedanti-γDPGA antibody or fragment thereof (for example, amounts that areeffective to alleviate one or more symptoms of a B. anthracisinfection).

The appropriate dose will vary depending on the characteristics of thesubject, for example, whether the subject is a human or non-human, theage, weight, and other health considerations pertaining to the conditionor status of the subject, the mode, route of administration, and numberof doses, and whether the pharmaceutical composition includes both adisclosed anti-γDPGA antibody or fragment thereof alone or inconjunction with one or more isolated anti anthrax toxin antibodies orfragment thereof or fragment thereof, such as one or more of an anti-PA,-LF and -EF antibody or fragment thereof, time and route ofadministration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the therapeuticcompositions for eliciting the desired activity or biological responsein the subject. Dosage regimens can be adjusted to provide an optimumprophylactic or therapeutic response. A therapeutically effective amountis also one in which any toxic or detrimental side effects of thecompound and/or other biologically active agent is outweighed inclinical terms by therapeutically beneficial effects. A non-limitingrange for a therapeutically effective amount of a disclosed anti-γDPGAantibody or fragment thereof and/or other biologically active agentwithin the methods and formulations of the disclosure is about 0.01mg/kg body weight to about 100 mg/kg body weight, such as about 0.05mg/kg to about 5 mg/kg body weight, or about 0.2 mg/kg to about 2 mg/kgbody weight or greater.

Therapeutic compositions that include a disclosed therapeutic agent canbe delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201, 1987; Buchwald et al., Surgery 88:507, 1980; Saudeket al., N. Engl. J. Med. 321:574, 1989) or by continuous subcutaneousinfusions, for example, using a mini-pump. An intravenous bag solutioncan also be employed. One factor in selecting an appropriate dose is theresult obtained, as measured by the methods disclosed here, as aredeemed appropriate by the practitioner. Other controlled release systemsare discussed in Langer (Science 249:1527-33, 1990).

In one example, a pump is implanted (for example see U.S. Pat. Nos.6,436,091; 5,939,380; and 5,993,414) Implantable drug infusion devicesare used to provide patients with a constant and long-term dosage orinfusion of a therapeutic agent. Such device can be categorized aseither active or passive.

Active drug or programmable infusion devices feature a pump or ametering system to deliver the agent into the patient's system. Anexample of such an active infusion device currently available is theMedtronic SYNCHROMED™ programmable pump. Passive infusion devices, incontrast, do not feature a pump, but rather rely upon a pressurized drugreservoir to deliver the agent of interest. An example of such a deviceincludes the Medtronic ISOMED™.

In particular examples, therapeutic compositions including a disclosedtherapeutic agent are administered by sustained-release systems.Suitable examples of sustained-release systems include suitablepolymeric materials (such as, semi-permeable polymer matrices in theform of shaped articles, for example films, or mirocapsules), suitablehydrophobic materials (for example as an emulsion in an acceptable oil)or ion exchange resins, and sparingly soluble derivatives (such as, forexample, a sparingly soluble salt). Sustained-release compositions canbe administered orally, parenterally, intracistemally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), or as an oral or nasal spray. Sustained-releasematrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481),copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman etal., Biopolymers 22:547-556, 1983, poly(2-hydroxyethyl methacrylate));(Langer et al., J. Biomed. Mater. Res. 15:167-277, 1981; Langer, Chem.Tech. 12:98-105, 1982, ethylene vinyl acetate (Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec, J. Parent. Sci. Tech. 44(2):58,1990). Alternatively, hydroxyapatite has been used as a microcarrier forcontrolled release of proteins (Ijntema et al., Int. J. Pharm. 112:215,1994). In yet another aspect, liposomes are used for controlled releaseas well as drug targeting of the lipid-capsulated drug (Betageri et al.,Liposome Drug Delivery Systems, Technomic Publishing Co., Inc.,Lancaster, Pa., 1993). Numerous additional systems for controlleddelivery of therapeutic proteins are known (for example, U.S. Pat. Nos.5,055,303; 5,188,837; 4,235,871; 4,501,728; 4,837,028; 4,957,735; and5,019,369; 5,055,303; 5,514,670; 5,413,797; 5,268,164; 5,004,697;4,902,505; 5,506,206; 5,271,961; 5,254,342; and 5,534,496).

The pharmaceutical compositions (medicaments) can be prepared for use inprophylactic regimens and administered to human or non-human subjects toprotect against infection by B. anthracis. Thus, the pharmaceuticalcompositions typically contain a pharmaceutically effective amount of adisclosed anti-γDPGA antibody or fragment thereof and optionally apharmaceutically effective amount of a complement protein or a fragmentthereof. In some cases the compositions are administered followinginfection, for example to treat the infection an increase B. anthracisclearance, in such applications, the pharmaceutical composition isadministered in a therapeutically effective amount. A therapeuticallyeffective amount is a quantity of a composition used to achieve adesired effect in a subject. For instance, this can be the amount of thecomposition necessary to inhibit infection by B. anthracis, to increaseB. anthracis clearance from the subject or to prevent or measurablyalter outward symptoms of B. anthracis infection from a subject. Whenadministered to a subject, a dosage will generally be used that willachieve target tissue concentrations that has been shown to achieve anin vitro or in vivo effect.

D. Diagnostic Methods and Kits

Methods are provided herein for the detection B. anthracis in vitro orin vivo for example in a biological sample obtained from a subject. Insome examples, detecting the presence of B. anthracis in a sampleindicates that the subject from which the sample was obtained has a B.anthracis infection. The sample can be any sample, including, but notlimited to, tissue from biopsies, autopsies and pathology specimens.Biological samples also include sections of tissues, for example, frozensections taken for histological purposes. Biological samples furtherinclude body fluids, such as blood, serum, plasma, sputum, spinal fluidor urine. A biological sample is typically obtained from a mammal, suchas a rat, mouse, cow, dog, guinea pig, rabbit, or primate. In someembodiments, the primate is macaque, chimpanzee, or a human.

In some examples, a sample is an environmental sample, such as a sampleobtained by swabbing a surface. Detection of B. anthracis in anenvironmental sample indicates that the environment from witch thesample is obtained is contaminated with B. anthracis.

The disclosed method includes contacting a sample, such as biologicalsample or environmental sample, with one or more of the disclosedanti-γDPGA antibodies or fragments thereof under conditions conductiveto the formation of an immune complex between the γDPGA present on thesurface of B. anthracis and the anti-γDPGA antibodies or fragmentsthereof, and detecting the immune complex, to detect the B. anthracisthe sample.

In one embodiment, a disclosed anti-γDPGA antibody or fragment thereofis directly labeled with a detectable label. In another embodiment, thedisclosed anti-γDPGA antibody or fragment thereof (the first antibody)is unlabeled and a second antibody or other molecule that can bind the adisclosed anti-γDPGA antibody or fragment thereof is labeled. As is wellknown to one of skill in the art, a second antibody is chosen that isable to specifically bind the specific species and class of the firstantibody. For example, if the first antibody is an IgG, then thesecondary antibody may be an anti-IgG. Other molecules that can bind toantibodies include, without limitation, Protein A and Protein G, both ofwhich are available commercially.

Suitable labels for the antibody or secondary antibody are describedabove, and include various enzymes, prosthetic groups, fluorescentmaterials, luminescent materials, magnetic agents and radioactivematerials. Non-limiting examples of suitable enzymes include horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase. Non-limiting examples of suitable prosthetic groupcomplexes include streptavidin/biotin and avidin/biotin. Non-limitingexamples of suitable fluorescent materials include umbelliferone,fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin. Anon-limiting exemplary luminescent material is luminol; a non-limitingexemplary a magnetic agent is gadolinium, and non-limiting exemplaryradioactive labels include ¹²⁵I, ¹³¹I, ³⁵S or ³H.

In other embodiments, B. anthracis is assayed in a sample by acompetition immunoassay utilizing B. anthracis standards, such as γDPGAstandards labeled with a detectable substance and a disclosed anti-γDPGAantibody or fragment thereof. In this assay, the sample, the labeled B.anthracis standards and the anti-γDPGA antibody or fragment thereof arecombined and the amount of labeled B. anthracis standard bound to theunlabeled antibody is determined. The amount of B. anthracis in thebiological sample is inversely proportional to the amount of labeled B.anthracis standard bound to the anti-γDPGA antibody or fragment thereof.

The immunoassays and method disclosed herein can be used for a number ofpurposes. In one embodiment, a disclosed anti-γDPGA antibody or fragmentthereof can be used to detect the amount of B. anthracis in a biologicalsample.

In some embodiments, a kit is provided for detecting B. anthracis in asample, such as a biological sample or an environmental sample. Kits fordetecting a B. anthracis will typically contain one or more of thedisclosed anti-γDPGA antibodies or fragment thereof, such as any of theantibodies or fragments disclosed herein. In some embodiments, anantibody fragment, such as an Fv fragment is included in the kit. For invivo uses, the antibody can be a scFv fragment. In a further embodiment,the antibody is labeled (for example, with a fluorescent, radioactive,or an enzymatic label).

In some embodiments, a kit additionally includes instructionalmaterials. The instructional materials may be written, in an electronicform (such as a computer diskette or compact disk) or may be visual(such as video files). The kits may also include additional componentsto facilitate the particular application for which the kit is designed.Thus, for example, the kit may additionally contain means of detecting alabel (such as enzyme substrates for enzymatic labels, filter sets todetect fluorescent labels, appropriate secondary labels such as asecondary antibody, or the like). The kits may additionally includebuffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting B. anthracis in a sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toγDPGA on the surface of B. anthracis. The antibody is allowed tospecifically bind under immunologically reactive conditions to form animmune complex, and the presence of the immune complex (bound antibody)is detected directly or indirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, magneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs. The antibodies can also beutilized in immunoassays such as but not limited to radioimmunoassays(RIAs), enzyme linked immunosorbant assays (ELISA), orimmunohistochemical assays. The antibodies can also be used forfluorescence activated cell sorting (FACS). A FACS employs a pluralityof color channels, low angle and obtuse light-scattering detectionchannels, and impedance channels, among other more sophisticated levelsof detection, to separate or sort cells (see U.S. Pat. No. 5,061,620).Thus, the disclosed antibodies or fragments thereof can be used in aconventional immunoassay, including, without limitation, an ELISA, anRIA, FACS, tissue immunohistochemistry, Western blot orimmunoprecipitation.

EXAMPLES Example 1 Isolation and Characterization of Poly-γ-D-GlutamicAcid Specific Fabs

Peptides (10 or 15 amino acids long) representing a fragment ofpoly-γ-D-glutamic acid (γDPGA) capsule of B. anthracis were conjugatedto either B. anthracis recombinant protective antigen (rPA) or tetanustoxoid (TT) and the conjugates were used to immunize chimpanzees.Fab-encoding gene fragments were amplified from the cDNA of chimpanzeebone marrow-derived mononuclear cells and cloned into pComb3H vector.The Fab-displaying phage library was panned against PGA and specificphage clones were recovered. DNA sequencing of the variable regions ofheavy (VH) and light (VL) chains from PGA-specific clones showed thatthere were two distinct clones. These two clones were designated 4C and11D. The 4C and 11D VH and VL amino acid sequences are shown in FIGS. 1Aand 1B.

The Fab sequences were converted into full-length IgG with human γ1constant regions and the IgGs were examined for their bindingspecificity by ELISA. Two anti-γDPGA monoclonal antibodies are specificto γDPGA as the monoclonal antibodies bound to γDPGA, but not tounrelated proteins (BSA, thyroglobulin, phosphorylase b, lysozyme, andcytochrome-c) (FIG. 2).

Example 2 Binding Affinity

This example shows the affinity measurement of the disclosed monoclonalantibodies for γDPGA. The kinetic analysis of the anti-γDPGA mAb wasperformed in a BIAcore 1000 (GE healthcare, Piscataway, N.J.). 4C or 11DIgG was immobilized onto a CM5 chip usingN-hydroxysuccinimide/1-ethyl-3-(3-dimethylaminopropyl)-carbodiimidecoupling chemistry. Two-fold serial dilutions of synthetic 10-merpeptide of r-D-glutamic acids from 20 nM to 0.078 nM were injected ontothe chip surface. The kinetic interaction between γDPGA peptide and 4Cand 11D IgG was displayed in the sensorgram and evaluated byBIAevaluation software using 1:1 (Langmuir) binding model. The resultshowed that the two anti-γDPGA monoclonal antibodies had high affinitiesfor the antigen (Table 2).

TABLE 2 Binding affinities of anti-γDPGA mAbs mAb K_(on) (M⁻¹s⁻¹)k_(off) (s⁻¹) K_(d) (nM)  4C 4.2 × 10⁶  2.3 × 10⁻³ 0.55  11D 1.1 × 10⁶9.04 × 10⁻⁵ 0.082

Example 3 Capsular Quellung Type Reaction

The capsular quellung type reaction was examined for the 4C monoclonalantibody. The Quellung reaction is a biochemical reaction in whichantibodies bind to the capsule of Streptococcus pneumoniae, Klebsiellapneumoniae, Neisseria meningitidis and Haemophilus influenzae and thusallow them to be visualized under a microscope. If the reaction ispositive, the capsule becomes opaque and appears to enlarge. Quellung isthe German word for “swelling” and describes the microscopic appearanceof pneumococcal or other bacterial capsules after their polysaccharideantigen has combined with a specific antibody. As a result of thiscombination, and precipitation of the large, complex molecule formed,the capsule appears to swell, because of increased surface tension, andits outlines become clearly demarcated.

Cells of formalin-killed B. anthracis (Ames 34 strain) were incubatedwith IgG1 of anti-γDPGA 4C monoclonal antibody at concentrations of 25μg/ml, 50 μg/ml and 100 μg/ml. The Fab of anti-γDPGA or IgG1 of anti-PAat 100 μg/ml were used as controls. The reactions were assessed by DICmicroscopy. The results (FIG. 3) showed that IgG1 of anti-γDPGA atconcentrations of 50 μg/ml and 100 μg/ml produced a rim type reaction atthe capsule perimeter. The reaction is specific for the anti-γDPGAmonoclonal antibody since anti-PA IgG1 at 100 μg/ml did not produce asimilar reaction. The reaction was also not observed when a lowerconcentration (25 μg/ml) of IgG1 or monovalent Fab of anti-γDPGA at ahigh concentration (100 μg/ml) was used.

Example 4 Opsonophagocytic Activity of Anti-γDPGA Monoclonal Antibodies

This example shows the measurement of opsonophagocytic activity of the4C and 11D anti-γDPGA monoclonal antibodies. The opsonophagocyticactivity of anti-γDPGA monoclonal antibodies was measured by theirability to kill B. anthracis cells in the presence of humanpolymorphonuclear leukocytes and complement. Opsonophagocytosis wasdefined as ≧50% killing compared with growth in control (no antibody)wells. The results showed that both anti-γDPGA mAbs had opsonophagocyticactivity (FIG. 4).

Example 5 Protection of Mice Against Challenge with Virulent AnthraxSpores

This example shows that the 4C and 11D anti-DPGA monoclonal antibodiesprovide efficient protection of mice against challenge with virulentanthrax spores. IgG1(hG1) and IgG3 (hG3) isotypes for each of the 4C and11D monoclonal antibodies were generated and the doses of 3 mg, 1 mg or0.3 mg of the antibodies were administered intraperitoneally into BALB/cmice. Eight mice were used for each dose group. The mice were infectedintra-tracheally with 1.48×10⁴ B. anthracis (Ames strain) spores atapproximately 18 hours post-monoclonal antibody treatment. The survivalwas monitored twice daily for 2 weeks post challenge.

The results showed that all monoclonal antibody-treated groups of micewere completely or partially protected from anthrax infection (Table 3).A comparison of the survival between monoclonal antibody treatments atthe lowest dose (0.3 mg/mouse) was statistically evaluated by theKaplan-Meier and the Logrank (Mantel-Cox) test (GraphPad Prism, Version4.0, San Diego, Calif.), as shown in FIG. 5. All monoclonalantibody-treated groups at the 0.3 mg/moue dose showed a significantincrease in survival when compared to the vehicle-treated group(p≦0.001). A higher percent survival was also observed with the IgG1isotype of both the 11D and the 4C monoclonal antibodies when comparedto their respective IgG3 isotype monoclonal antibodies at the 0.3mg/mouse dose, although the difference was not statistically significantby the Logrank test.

TABLE 3 Survival Following an Intratracheal Challenge with B. anthracis(Ames strain) Spores in mAb Pre-Treated BALB/c Mice mAb Treatment mAbNumber of Mice Alive/Day Post-Infection^(d) Percent Group^(a) dose/mouseDay 0^(b) Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 9 Day 13 Day 15Survival Vehicle None 8 0 0 0 0 0 0 0 0 0 0 Control (DPBS)^(e) CH11D- 3mg 8 8 8 8 8 8 8 7 6 6 75 hG3 1 mg 8 8 8 8 8 8 8 8 8 8 100 0.3 mg  7^(c) 6 5 5 5 5 5 4 4 4 57 CH11D- 3 mg 7^(c) 7 7 7 7 7 7 7 7 7 100 hG1 1mg 7^(c) 7 7 7 7 7 7 7 7 7 100 0.3 mg   7^(c) 7 7 7 6 6 6 6 6 6 86 CH4C-3 mg 5^(c) 5 4 4 4 4 4 4 4 4 80 hG3 1 mg 7^(c) 7 6 6 5 5 5 5 5 5 71 0.3mg   8 8 8 7 7 6 5 5 5 5 63 CH4C- 3 mg 8 8 8 8 8 8 8 8 8 8 100 hG1 1 mg8 8 8 8 8 8 8 8 8 8 100 0.3 mg   8 8 8 8 8 8 8 8 8 8 100 ^(a)The micewere treated with the designated dose of IgG1 (hG1) or IgG3 (hg3)isotypes of CH11D or CH4C mAbs. ^(b)18-24 h post-treatment with mAbs,the mice were infected intratracheally with 1.48 × 10⁴ B. anthracisspores/mouse. ^(c)All groups started with 8 mice/group, but a few werelost during the inoculation procedure due to anesthesia complications.^(d)The mice were monitored twice daily. The data indicate the day achange occurred. ^(e)1 X Dulbecco's PBS.

The data demonstrate that pre-treatment with the anti-capsularmonoclonal antibodies 11D or 4C, at doses as low as 0.3 mg/mouse,provided significant protection against a lethal pulmonary challengewith B. anthracis (Ames strain) spores in BALB/c mice and IgG1 was moreeffective than IgG3 for both monoclonal antibodies.

Example 6 Mouse Model of Anti-γDPGA Protection from Lethal Challengewith Bacillus anthracis

This example describes exemplary procedures for testing the efficacy ofanti-γDPGA antibodies to treat Bacillus anthracis infections. Using themouse model of Bacillus anthracis infection developed by Lyons et al.(Infect Immun. 72(8): 4801-4809, 2004) the efficacy of the monoclonalantibodies 11D and 4C is tested.

Briefly, B. anthracis (Ames strain) spore stock is streaked onto a bloodagar plate and incubated overnight at 37° C. Prewarmed 2×SG medium isthen inoculated with one colony from the overnight plate culture andincubated at 37° C. on a shaker. A sample from this preculture (1.25 ml)is added to 50 ml of 2×SG medium (prewarmed) in a 500-ml filter-topplastic flask and shaken for 24 h. At the end of the 24-hour period, 200ml of sterile double-distilled H₂O is added to the culture, andincubation is continued with shaking for 40 more hours. After the40-hour incubation, the sporulation of the culture is evaluated. Theculture is then transferred to a sterile centrifuge at 4 to 10° C. for20 min, washed one time with sterile phosphate-buffered saline (PBS),and resuspended in 50 ml of sterile PBS. The culture is heated to 68° C.for 40 minutes prior to further preparation to remove potentiallycontaminating vegetative cells. Aliquots of the final solution aretransferred into vials and frozen at −80° C. The titer of the stock isdetermined by plating serial dilutions onto blood agar plates.

An aliquot of the working stock is removed from the freezer and thawedto room temperature in a biohazard hood. The stock is then diluted inserial dilutions with sterile PBS to a desired concentration.

BALB/c mice between 6 and 8 weeks of age are inoculated and kept in ananimal biosafety level 3 containment area. For intranasal (i.n.)inoculations, mice are lightly anesthetized with isoflurane, and a 50-μlinoculum is placed on the nares for inhalation into the lungs. For intratrachea (i.t.) inoculations, mice are anesthetized with avertin andrestrained on a small board, a small incision is made through the skinover the trachea, and the underlying tissue is separated. A bent30-gauge needle attached by sterile polypropylene tubing to a tuberculinsyringe is used; the needle is inserted into and parallel with thetrachea, and a 50-μl inoculum is delivered into the lung. Forsubcutaneous (s.c.) inoculation, doses of spores are delivered in atotal volume of 200 μl of PBS using a 29-gauge needle attached to aninsulin syringe.

Mice are administered a therapeutic amount of anti-γDPGA antibody. Theanti-γDPGA antibody can be administered at doses of 1 μg/kg body weightto about 1 mg/kg body weight per dose, such as 1 μg/kg body weight-100μg/kg body weight per dose, 100 μg/kg body weight-500 μg/kg body weightper dose, or 500 μg/kg body weight-1000 μg/kg body weight per dose oreven greater. The agent can be administered in several doses, forexample continuously, daily, weekly, or monthly. In some examples, themice are administered complement protein or a fragment there of todetermine the affects of complement on B. anthracis infection viacoadministration with the disclosed anti-γDPGA antibodies.

After administration of anti-γDPGA antibody, mice are monitored forsigns of disease, for example, from visual inspection and determinationof bacterial counts.

Example 7 Treatment of Subjects

This example describes methods that can be used to treat a subject thathas or is at risk of having an infection from B. anthracis.

In some examples, one or more anti-γDPGA antibodies disclosed herein isadministered alone or in combination with one or more isolatedanti-anthrax toxin antibodies. or a fragment thereof, such as one ormore of an anti-PA, -LF or -EF antibody or a fragment thereof. Inparticular examples, the method includes screening a subject having,thought to have, or at risk of having (for example due to impairedimmunity, physiological status, or exposure to a B. anthracis) a B.anthracis infection. Subjects of an unknown infection status can beexamined to determine if they have an infection, for example usingserological tests, physical examination, enzyme-linked immunosorbentassay (ELISA), radiological screening or other diagnostic techniqueknown to those of skill in the art. Subjects found to (or known to) havea B. anthracis infection and thereby treatable by administration of oneor more of the disclosed anti-γDPGA antibodies are selected to receiveanti-γDPGA antibody and optionally one or more isolated anti-anthraxtoxin antibodies or fragment thereof, such as one or more of an anti-PA,-LF or -EF antibody or fragment thereof.

Subjects selected for treatment can be administered a therapeutic amountof anti-γDPGA antibody alone or in combination with a therapeutic amountof one or more isolated anti-anthrax toxin antibodies or fragmentthereof, such as one or more of an anti-PA, -LF or -EF antibody orfragment thereof. The antibody can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight-100 μg/kg body weight per dose, 100 μg/kg body weight-500 μg/kgbody weight per dose, or 500 μg/kg body weight-1000 μg/kg body weightper dose or even greater. However, the particular dose can be determinedby a skilled clinician. The agent can be administered in several doses,for example continuously, daily, weekly, or monthly.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

Subjects selected for treatment can also be administered a therapeuticamount of complement protein or a fragment thereof. The complementprotein or a fragment thereof can be administered at doses of 1 μg/kgbody weight to about 1 mg/kg body weight per dose, such as 1 μg/kg bodyweight-100 μg/kg body weight per dose, 100 μg/kg body weight-500 μg/kgbody weight per dose, or 500 μg/kg body weight-1000 μg/kg body weightper dose. However, the particular dose can be determined by a skilledclinician. The one or more isolated anti-anthrax toxin antibodies orfragment thereof or fragment thereof, such as one or more of an anti-PA,-LF and -EF antibody or fragment thereof, can be administeredconcurrently or sequentially with the anti-γDPGA antibody. Theanti-γDPGA antibody and/or the one or more isolated anti-anthrax toxinantibodies or fragment thereof, such as one or more of an anti-PA, -LFor -EF antibody or fragment thereof, can be administered in one orseveral doses, for example continuously, daily, weekly, or monthly. Whenadministered sequentially the time separating the administration of theanti-γDPGA antibody and one or more isolated anti-anthrax toxinantibodies or fragment thereof, such as one or more of an anti-PA, -LFor -EF antibody or fragment thereof, can be seconds, minutes, hours,days, or even weeks.

The mode of administration can be any used in the art. The amount ofagent administered to the subject can be determined by a clinician, andmay depend on the particular subject treated. Specific exemplary amountsare provided herein (but the disclosure is not limited to such doses).

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used, andit is intended that the disclosure may be practiced otherwise than asspecifically described herein. Features, characteristics, compounds,chemical moieties, or examples described in conjunction with aparticular aspect, embodiment, or example of the invention are to beunderstood to be applicable to any other aspect, embodiment, or exampleof the invention. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims.

We claim:
 1. A method for protecting a subject from a Bacillus anthracisinfection, comprising: selecting a subject with a Bacillus anthracisinfection or suspected of having a Bacillus anthracis infection; andadministering to the subject a therapeutically effective amount of apharmaceutical composition comprising an isolated monoclonal antibody orantigen binding fragment thereof and a pharmaceutically acceptablecarrier, wherein the monoclonal antibody or antigen binding fragmentcomprises a heavy chain (H) with a H-complementarity determining region(CDR)1, H-CDR2 and H-CDR3 region and a light chain (L) with a L-CDR1,L-CDR2 and L-CDR3 region, wherein: (a) the heavy chain of the monoclonalantibody or antigen binding fragment comprises the H-CDR1, H-CDR2 andH-CDR3 of SEQ ID NO: 3 and the light chain of the monoclonal antibody orantigen binding fragment comprises the L-CDR1, L-CDR2 and L-CDR3 of SEQID NO: 4; or (b) the heavy chain of the monoclonal antibody or antigenbinding fragment comprises the H-CDR1, H-CDR2 and H-CDR3 of SEQ ID NO: 5and the light chain of the monoclonal antibody or antigen bindingfragment comprises the L-CDR1, L-CDR2 and L-CDR3 of SEQ ID NO: 6, andwherein the antibody or antigen binding fragment specifically bindspoly-γ-D-glutamic acid (γDPGA), thereby protecting the subject from theBacillus anthracis infection.
 2. The method of claim 1, wherein (a) theH-CDR1 comprises the amino acid sequence set forth as amino acids 28 to32 of SEQ ID NO: 3, the H-CDR2 comprises the amino acid sequence setforth as amino acids 47 to 55 of SEQ ID NO: 3, and the H-CDR3 comprisesthe amino acid sequence set forth as amino acids 95 to 111 of SEQ ID NO:3, and wherein the antibody has a light chain with a L-CDR 1, L- CDR2and L-CDR3 region, and wherein L-CDR1 comprises the amino acid sequenceset forth as amino acids 23 to 33 of SEQ ID NO: 4, L-CDR2 comprises theamino acid sequence set forth as amino acids 48 to 55 of SEQ ID NO: 4,and L-CDR3 comprises the amino acid sequence set forth as amino acids 88to 96 of SEQ ID NO: 4; or (b) wherein H-CDR1 comprises the amino acidsequence set forth as amino acids 27 to 31 of SEQ ID NO: 5, H-CDR2comprises the amino acid sequence set forth as amino acids 46 to 54 ofSEQ ID NO: 5, and H-CDR3 comprises the amino acid sequence set forth asamino acids 94 to 110 of SEQ ID NO: 5 and wherein the antibody has lightchain with a L-CDR1, L-CDR2 and L-CDR3 region, and wherein L-CDR1comprises the amino acid sequence set forth as amino acids 22 to 32 ofSEQ ID NO: 6, L-CDR2 comprises the amino acid sequence set forth asamino acids 47 to 54 of SEQ ID NO: 6, and L-CDR3 comprises the aminoacid sequence set forth as amino acids 87 to 95 of SEQ ID NO:
 6. 3. Themethod of claim 2, wherein: (a) the heavy chain comprises the amino acidsequence according to SEQ ID NO. 3; and the light chain comprises theamino acid sequence according to SEQ ID NO. 4; or (b) the heavy chaincomprises the amino acid sequence according to SEQ ID NO. 5; and thelight chain comprises the amino acid sequence according to SEQ ID NO. 6.4. The method of claim 2, further comprising, administering to thesubject a therapeutically effective amount of one or more isolatedanti-anthrax toxin antibodies or a fragment thereof.
 5. The method ofclaim 4, wherein the one or more isolated anti-anthrax toxin antibodiesor a fragment thereof comprises one or more of an anti-protectiveantigen (PA), -lethal factor (LF), or -edema factor (EF) antibody or afragment thereof.
 6. The method of claim 2, wherein protecting thesubject from the Bacillus anthracis infection comprises protecting thesubject from a subsequent challenge with Bacillus anthracis spores. 7.The method of claim 2, wherein the antigen binding fragment is a Fab′fragment, a F(ab)′₂ fragment, a single chain Fv protein (scFv), or adisulfide stabilized Fv protein (dsFv).
 8. The method of claim 6,wherein the antibody is an IgG.
 9. The method of claim 8, wherein theantibody comprises a human γ1 constant region.
 10. The method of claim6, wherein the antibody or antigen binding fragment is humanized. 11.The method of claim 6, wherein the pharmaceutical composition isadministered to the subject after exposure to Bacillus anthracis. 12.The method of claim 6, wherein the subject is a human.
 13. The method ofclaim 6, wherein the pharmaceutical composition is administeredintramuscularly.
 14. The method of claim 7, wherein (a) the H-CDR1comprises the amino acid sequence set forth as amino acids 28 to 32 ofSEQ ID NO: 3, the H-CDR2 comprises the amino acid sequence set forth asamino acids 47 to 55 of SEQ ID NO: 3, and the H-CDR3 comprises the aminoacid sequence set forth as amino acids 95 to 111 of SEQ ID NO: 3, andwherein the antibody has a light chain with a L-CDR1, L-CDR2 and L-CDR3region, and wherein L-CDR1 comprises the amino acid sequence set forthas amino acids 23 to 33 of SEQ ID NO: 4, L-CDR2 comprises the amino acidsequence set forth as amino acids 48 to 55 of SEQ ID NO: 4, and L-CDR3comprises the amino acid sequence set forth as amino acids 88 to 96 ofSEQ ID NO:
 4. 15. The method of claim 7 wherein H-CDR1 comprises theamino acid sequence set forth as amino acids 27 to 31 of SEQ ID NO: 5,H-CDR2 comprises the amino acid sequence set forth as amino acids 46 to54 of SEQ ID NO: 5, and H-CDR3 comprises the amino acid sequence setforth as amino acids 94 to 110 of SEQ ID NO: 5 and wherein the antibodyhas light chain with a L-CDR1, L-CDR2 and L-CDR3 region, and whereinL-CDR1 comprises the amino acid sequence set forth as amino acids 22 to32 of SEQ ID NO: 6, L-CDR2 comprises the amino acid sequence set forthas amino acids 47 to 54 of SEQ ID NO: 6, and L-CDR3 comprises the aminoacid sequence set forth as amino acids 87 to 95 of SEQ ID NO: 6.